Although glycogen synthase kinase β (Gsk3β) has been shown to regulate tissue inflammation, whether and how it regulates inflammation resolution versus inflammation activation is unclear. In a murine liver, partial warm ischemia/reperfusion injury (IRI) model, we found that Gsk3β inhibitory phosphorylation increased at both the early-activation and late-resolution stages of the disease. Myeloid Gsk3β deficiency not only alleviated liver injuries, it also facilitated the restoration of liver homeostasis. Depletion of Kupffer cells prior to the onset of liver ischemia diminished the differences between the WT and Gsk3β-KO mice in the activation of liver IRI. However, the resolution of liver IRI remained accelerated in Gsk3β-KO mice. In CD11b-DTR mice, Gsk3β-deficient BM-derived macrophages (BMMs) facilitated the resolution of liver IRI as compared with WT cells. Furthermore, Gsk3β deficiency promoted the reparative phenotype differentiation in vivo in liver-infiltrating macrophages and in vitro in BMMs. Gsk3 pharmacological inhibition promoted the resolution of liver IRI in WT, but not myeloid MerTK-deficient, mice. Thus, Gsk3β regulates liver IRI at both activation and resolution stages of the disease. Gsk3 inactivation enhances the proresolving function of liver-infiltrating macrophages in an MerTK-dependent manner.
Hanwen Zhang, Ming Ni, Han Wang, Jing Zhang, Dan Jin, Ronald W. Busuttil, Jerzy W. Kupiec-Weglinski, Wei Li, Xuehao Wang, Yuan Zhai
Carbohydrate response element–binding protein (ChREBP) is a carbohydrate-sensing transcription factor that regulates both adaptive and maladaptive genomic responses in coordination of systemic fuel homeostasis. Genetic variants in the ChREBP locus associate with diverse metabolic traits in humans, including circulating lipids. To identify novel ChREBP-regulated hepatokines that contribute to its systemic metabolic effects, we integrated ChREBP ChIP-Seq analysis in mouse liver with human genetic and genomic data for lipid traits and identified hepatocyte growth factor activator (HGFAC) as a promising ChREBP-regulated candidate in mice and humans. HGFAC is a protease that activates the pleiotropic hormone hepatocyte growth factor. We demonstrate that HGFAC-KO mice had phenotypes concordant with putative loss-of-function variants in human HGFAC. Moreover, in gain- and loss-of-function genetic mouse models, we demonstrate that HGFAC enhanced lipid and glucose homeostasis, which may be mediated in part through actions to activate hepatic PPARγ activity. Together, our studies show that ChREBP mediated an adaptive response to overnutrition via activation of HGFAC in the liver to preserve glucose and lipid homeostasis.
Ashot Sargsyan, Ludivine Doridot, Sarah A. Hannou, Wenxin Tong, Harini Srinivasan, Rachael Ivison, Ruby Monn, Henry H. Kou, Jonathan M. Haldeman, Michelle Arlotto, Phillip J. White, Paul A. Grimsrud, Inna Astapova, Linus T. Tsai, Mark A. Herman
A role of CD4+ T cells during the progression from nonalcoholic fatty liver disease (NAFLD) to nonalcoholic steatohepatitis (NASH) has been suggested, but which polarization state of these cells characterizes this progression and the development of fibrosis remain unclear. In addition, a gut-liver axis has been suggested to play a role in NASH, but the role of CD4+ T cells in this axis has just begun to be investigated. Combining single-cell RNA sequencing and multiple-parameter flow cytometry, we provide the first cell atlas to our knowledge focused on liver-infiltrating CD4+ T cells in patients with NAFLD and NASH, showing that NASH is characterized by a population of multicytokine-producing CD4+ T cells. Among these cells, only those with a Th17 polarization state were enriched in patients with advanced fibrosis. In parallel, we observed that Bacteroides appeared to be enriched in the intestine of NASH patients and to correlate with the frequency of multicytokine-producing CD4+ T cells. In short, we deliver a CD4+ T cell atlas of NAFLD and NASH, providing the rationale to target CD4+ T cells with a Th17 polarization state to block fibrosis development. Finally, our data offer an early indication to test whether multicytokine-producing CD4+ T cells are part of the gut-liver axis characterizing NASH.
Anna Woestemeier, Pasquale Scognamiglio, Yu Zhao, Jonas Wagner, Franziska Muscate, Christian Casar, Francesco Siracusa, Filippo Cortesi, Theodora Agalioti, Simone Müller, Adrian Sagebiel, Leonie Konczalla, Ramez Wahib, Karl-Frederick Karstens, Anastasios D. Giannou, Anna Duprée, Stefan Wolter, Milagros N. Wong, Anne K. Mühlig, Agata A. Bielecka, Vikas Bansal, Tianran Zhang, Oliver Mann, Victor G. Puelles, Tobias B. Huber, Ansgar W. Lohse, Jakob R. Izbicki, Noah W. Palm, Stefan Bonn, Samuel Huber, Nicola Gagliani
Chronic inflammation is associated with lung tumorigenesis, in which NF-κB–mediated epigenetic regulation plays a critical role. Lung tumor suppressor G protein–coupled receptor, family C, member 5A (GPRC5A), is repressed in most non–small cell lung cancer (NSCLC); however, the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induced GPRC5A repression both in vitro and in vivo. Intriguingly, transactivation of NF-κB downstream targets was not required, but the transactivation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in the GPRC5A promoter. Instead, p65 was complexed with retinoic acid receptor α/β (RARα/β) and recruited to the RA response element site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complexing and suppressed transcription. Notably, phosphorylation on serine 276 of p65 was required for interaction with RARα/β and repression of GPRC5A. Moreover, NF-κB–mediated epigenetic repression was through suppression of acetylated histone H3K9 (H3K9ac), but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a histone deacetylase inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via a complex with RARα/β and mediates epigenetic repression via suppression of H3K9ac.
Hongyong Song, Xiaofeng Ye, Yueling Liao, Siwei Zhang, Dongliang Xu, Shuangshuang Zhong, Bo Jing, Tong Wang, Beibei Sun, Jianhua Xu, Wenzheng Guo, Kaimi Li, Min Hu, Yanbin Kuang, Jing Ling, Tuo Zhang, Yadi Wu, Jing Du, Feng Yao, Y. Eugene Chin, Qi Wang, Binhua P. Zhou, Jiong Deng
Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability that exceeds genic effects. Dysregulated metabolism and immune cell function are key disease modifiers. The tryptophan metabolites, kynurenines, produced through indoleamine 2,3-dioxygenase 1 (IDO1), are known immunomodulators. Here, we study the role of tryptophan metabolism in PKD using an orthologous disease model (C57BL/6J Pkd1RC/RC). We found elevated kynurenine and IDO1 levels in Pkd1RC/RC kidneys versus wild type. Further, IDO1 levels were increased in ADPKD cell lines. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity, as measured by cystic index and percentage kidney weight normalized to body weight. Consistent with an immunomodulatory role of kynurenines, Pkd1RC/RC;Ido1–/– mice presented with significant changes in the cystic immune microenvironment (CME) versus controls. Kidney macrophage numbers decreased and CD8+ T cell numbers increased, both known PKD modulators. Also, pharmacological IDO1 inhibition in Pkd1RC/RC mice and kidney-specific Pkd2-knockout mice with rapidly progressive PKD resulted in less severe PKD versus controls, with changes in the CME similar to those in the genetic model. Our data suggest that tryptophan metabolism is dysregulated in ADPKD and that its inhibition results in changes to the CME and slows disease progression, making IDO1 a therapeutic target for ADPKD.
Dustin T. Nguyen, Emily K. Kleczko, Nidhi Dwivedi, Marie-Louise T. Monaghan, Berenice Y. Gitomer, Michel B. Chonchol, Eric T. Clambey, Raphael A. Nemenoff, Jelena Klawitter, Katharina Hopp
Pulmonary fibrosis is characterized by stiffening of the extracellular matrix. Fibroblasts migrate in the direction of greater stiffness, a phenomenon termed durotaxis. The mechanically guided fibroblast migration could be a crucial step in the progression of lung fibrosis. In this study, we found primary human lung fibroblasts sense increasing matrix stiffness with a change of mitochondrial dynamics in favor of mitochondrial fission and increased production of ATP. Mitochondria polarize in the direction of a physiologically relevant stiffness gradient, with conspicuous localization to the leading edge, primarily lamellipodia and filopodia, of migrating lung fibroblasts. Matrix stiffness–regulated mitochondrial fission and durotactic lung fibroblast migration are mediated by a dynamin-related protein 1/mitochondrial fission factor–dependent (DRP1/MFF-dependent) pathway. Importantly, we found that the DRP1/MFF pathway is activated in fibrotic lung myofibroblasts in both human IPF and bleomycin-induced mouse lung fibrosis. These findings suggest that energy-producing mitochondria need to be sectioned via fission and repositioned in durotactic lung fibroblasts to meet the higher energy demand. This represents a potentially new mechanism through which mitochondria may contribute to the progression of fibrotic lung diseases. Inhibition of durotactic migration of lung fibroblasts may play an important role in preventing the progression of human idiopathic pulmonary fibrosis.
Ting Guo, Chun-sun Jiang, Shan-Zhong Yang, Yi Zhu, Chao He, A. Brent Carter, Veena B. Antony, Hong Peng, Yong Zhou
Despite the efficacy of tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML), malignant long-term hematopoietic stem cells (LT-HSCs) persist as a source of relapse. However, LT-HSCs are heterogenous and the most primitive, drug-resistant LT-HSC subpopulations are not well characterized. In normal hematopoiesis, self-renewal and long-term reconstitution capacity are enriched within LT-HSCs with low c-Kit expression (c-KITlo). Here, using a transgenic CML mouse model, we found that long-term engraftment and leukemogenic capacity were restricted to c-KITlo CML LT-HSCs. CML LT-HSCs demonstrated enhanced differentiation with expansion of mature progeny following exposure to the c-KIT ligand, stem cell factor (SCF). Conversely, SCF deletion led to depletion of normal LT-HSCs but increase in c-KITlo and total CML LT-HSCs with reduced generation of mature myeloid cells. CML c-KITlo LT-HSCs showed reduced cell cycling and expressed enhanced quiescence and inflammatory gene signatures. SCF administration led to enhanced depletion of CML primitive progenitors but not LT-HSCs after TKI treatment. Human CML LT-HSCs with low or absent c-KIT expression were markedly enriched after TKI treatment. We conclude that CML LT-HSCs expressing low c-KIT levels are enriched for primitive, quiescent, drug-resistant leukemia-initiating cells and represent a critical target for eliminating disease persistence.
Mansi Shah, Harish Kumar, Shaowei Qiu, Hui Li, Mason Harris, Jianbo He, Ajay Abraham, David K. Crossman, Andrew Paterson, Robert S. Welner, Ravi Bhatia
We determined whether gut microbiota-produced trimethylamine (TMA) is oxidized into trimethylamine N-oxide (TMAO) in nonliver tissues and whether TMAO promotes inflammation via trained immunity (TI). We found that endoplasmic reticulum (ER) stress genes were coupregulated with MitoCarta genes in chronic kidney diseases (CKD); TMAO upregulated 190 genes in human aortic endothelial cells (HAECs); TMAO synthesis enzyme flavin-containing monooxygenase 3 (FMO3) was expressed in human and mouse aortas; TMAO transdifferentiated HAECs into innate immune cells; TMAO phosphorylated 12 kinases in cytosol via its receptor PERK and CREB, and integrated with PERK pathways; and PERK inhibitors suppressed TMAO-induced ICAM-1. TMAO upregulated 3 mitochondrial genes, downregulated inflammation inhibitor DARS2, and induced mitoROS, and mitoTEMPO inhibited TMAO-induced ICAM-1. β-Glucan priming, followed by TMAO restimulation, upregulated TNF-α by inducing metabolic reprogramming, and glycolysis inhibitor suppressed TMAO-induced ICAM-1. Our results have provided potentially novel insights regarding TMAO roles in inducing EC activation and innate immune transdifferentiation and inducing metabolic reprogramming and TI for enhanced vascular inflammation, and they have provided new therapeutic targets for treating cardiovascular diseases (CVD), CKD-promoted CVD, inflammation, transplantation, aging, and cancer.
Fatma Saaoud, Lu Liu, Keman Xu, Ramon Cueto, Ying Shao, Yifan Lu, Yu Sun, Nathaniel W. Snyder, Sheng Wu, Ling Yang, Yan Zhou, David L. Williams, Chuanfu Li, Laisel Martinez, Roberto I. Vazquez-Padron, Huaqing Zhao, Xiaohua Jiang, Hong Wang, Xiaofeng Yang
FOXD1+ cell–derived stromal cells give rise to pericytes and fibroblasts that support the kidney vasculature and interstitium but are also major precursors of myofibroblasts. ZEB2 is a SMAD-interacting transcription factor that is expressed in developing kidney stromal progenitors. Here we show that Zeb2 is essential for normal FOXD1+ stromal progenitor development. Specific conditional knockout of mouse Zeb2 in FOXD1+ stromal progenitors (Zeb2 cKO) leads to abnormal interstitial stromal cell development, differentiation, and kidney fibrosis. Immunofluorescent staining analyses revealed abnormal expression of interstitial stromal cell markers MEIS1/2/3, CDKN1C, and CSPG4 (NG2) in newborn and 3-week-old Zeb2-cKO mouse kidneys. Zeb2-deficient FOXD1+ stromal progenitors also took on a myofibroblast fate that led to kidney fibrosis and kidney failure. Cell marker studies further confirmed that these myofibroblasts expressed pericyte and resident fibroblast markers, including PDGFRβ, CSPG4, desmin, GLI1, and NT5E. Notably, increased interstitial collagen deposition associated with loss of Zeb2 in FOXD1+ stromal progenitors was accompanied by increased expression of activated SMAD1/5/8, SMAD2/3, SMAD4, and AXIN2. Thus, our study identifies a key role of ZEB2 in maintaining the cell fate of FOXD1+ stromal progenitors during kidney development, whereas loss of ZEB2 leads to differentiation of FOXD1+ stromal progenitors into myofibroblasts and kidney fibrosis.
Sudhir Kumar, Xueping Fan, Hila Milo Rasouly, Richa Sharma, David J. Salant, Weining Lu
BACKGROUND. To minimize COVID-19 pandemic burden and spread, 3-dose vaccination campaigns commenced worldwide. Since patients who are pregnant are at increased risk for severe disease, they were recently included in that policy, despite the lack of available evidence regarding the impact of a third boosting dose during pregnancy, underscoring the urgent need for relevant data. We aimed to characterize the effect of the third boosting dose of mRNA Pfizer BNT162b2 vaccine in pregnancy.METHODS. We performed a prospective cohort study of anti–SARS-CoV-2 antibody titers (n = 213) upon delivery in maternal and cord blood of naive fully vaccinated parturients who received a third dose (n = 86) as compared with 2-dose recipients (n = 127).RESULTS. We found a robust surge in maternal and cord blood levels of anti–SARS-CoV-2 titers at the time of delivery, when comparing pregnancies in which the mother received a third boosting dose with 2-dose recipients. The effect of the third boosting dose remained significant when controlling for the trimester of last exposure, suggesting additive immunity extends beyond that obtained after the second dose. Milder side effects were reported following the third dose, as compared with the second vaccine dose, among the fully vaccinated group.CONCLUSION. The third boosting dose of mRNA Pfizer BNT162b2 vaccine augmented maternal and neonatal immunity with mild side effects. These data provide evidence to bolster clinical and public health guidance, reassure patients, and increase vaccine uptake among patients who are pregnant.FUNDING. Israel Science Foundation KillCorona grant 3777/19; Research grant from the “Ofek” Program of the Hadassah Medical Center.
Adva Cahen-Peretz, Lilah Tsaitlin-Mor, Hadas Allouche Kam, Racheli Frenkel, Maor Kabessa, Sarah M. Cohen, Michal Lipschuetz, Esther Oiknine-Djian, Sapir Lianski, Debra Goldman-Wohl, Asnat Walfisch, Michal Kovo, Michal Neeman, Dana G. Wolf, Simcha Yagel, Ofer Beharier
Dysfunction of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, is implicated in pulmonary disease pathogenesis, highlighting the importance of human in vitro models. However, AEC2-like cells in culture have yet to be directly compared to their in vivo counterparts at single-cell resolution. Here, we performed head-to-head comparisons among the transcriptomes of primary (1°) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell–derived AEC2s (iAEC2s). We found each population occupied a distinct transcriptomic space with cultured AEC2s (1° and iAEC2s) exhibiting similarities to and differences from freshly purified 1° cells. Across each cell type, we found an inverse relationship between proliferative and maturation states, with preculture 1° AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2s did not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s cocultured with fibroblasts acquired a transitional cell state described in mice and humans to arise during fibrosis or following injury. Hence, we provide direct comparisons of the transcriptomic programs of 1° and engineered AEC2s, 2 in vitro models that can be harnessed to study human lung health and disease.
Konstantinos-Dionysios Alysandratos, Carolina Garcia-de-Alba, Changfu Yao, Patrizia Pessina, Jessie Huang, Carlos Villacorta-Martin, Olivia T. Hix, Kasey Minakin, Claire L. Burgess, Pushpinder Bawa, Aditi Murthy, Bindu Konda, Michael F. Beers, Barry R. Stripp, Carla F. Kim, Darrell N. Kotton
The central physiological role of the bone marrow renders bone marrow stromal cells (BMSCs) particularly sensitive to aging. With bone aging, BMSCs acquire a differentiation potential bias in favor of adipogenesis over osteogenesis, and the underlying molecular mechanisms remain unclear. Herein, we investigated the factors underlying age-related changes in the bone marrow and their roles in BMSCs’ differentiation. Antibody array revealed that CC chemokine ligand 3 (CCL3) accumulation occurred in the serum of naturally aged mice along with bone aging phenotypes, including bone loss, bone marrow adiposity, and imbalanced BMSC differentiation. In vivo Ccl3 deletion could rescue these phenotypes in aged mice. CCL3 improved the adipogenic differentiation potential of BMSCs, with a positive feedback loop between CCL3 and C/EBPα. CCL3 activated C/EBPα expression via STAT3, while C/EBPα activated CCL3 expression through direct promoter binding, facilitated by DNA hypomethylation. Moreover, CCL3 inhibited BMSCs’ osteogenic differentiation potential by blocking β-catenin activity mediated by ERK-activated Dickkopf-related protein 1 upregulation. Blocking CCL3 in vivo via neutralizing antibodies ameliorated trabecular bone loss and bone marrow adiposity in aged mice. This study provides insights regarding age-related bone loss and bone marrow adiposity pathogenesis and lays a foundation for the identification of new targets for senile osteoporosis treatment.
Degang Yu, Shuhong Zhang, Chao Ma, Sen Huang, Long Xu, Jun Liang, Huiwu Li, Qiming Fan, Guangwang Liu, Zanjing Zhai
Systemic iron metabolism is disrupted in chronic kidney disease (CKD). However, little is known about local kidney iron homeostasis and its role in kidney fibrosis. Kidney-specific effects of iron therapy in CKD also remain elusive. Here, we elucidate the role of macrophage iron status in kidney fibrosis and demonstrate that it is a potential therapeutic target. In CKD, kidney macrophages exhibited depletion of labile iron pool (LIP) and induction of transferrin receptor 1, indicating intracellular iron deficiency. Low LIP in kidney macrophages was associated with their defective antioxidant response and proinflammatory polarization. Repletion of LIP in kidney macrophages through knockout of ferritin heavy chain (Fth1) reduced oxidative stress and mitigated fibrosis. Similar to Fth1 knockout, iron dextran therapy, through replenishing macrophage LIP, reduced oxidative stress, decreased the production of proinflammatory cytokines, and alleviated kidney fibrosis. Interestingly, iron markedly decreased TGF-β expression and suppressed TGF-β–driven fibrotic response of macrophages. Iron dextran therapy and FtH suppression had an additive protective effect against fibrosis. Adoptive transfer of iron-loaded macrophages alleviated kidney fibrosis, validating the protective effect of iron-replete macrophages in CKD. Thus, targeting intracellular iron deficiency of kidney macrophages in CKD can serve as a therapeutic opportunity to mitigate disease progression.
Edwin Patino, Divya Bhatia, Steven Z. Vance, Ada Antypiuk, Rie Uni, Chantalle Campbell, Carlo G. Castillo, Shahd Jaouni, Francesca Vinchi, Mary E. Choi, Oleh Akchurin
Glucocorticoids remain a cornerstone of therapeutic regimes for autoimmune and chronic inflammatory diseases — for example, in different forms of crescentic glomerulonephritis — because of their rapid antiinflammatory effects, low cost, and wide availability. Despite their routine use for decades, the underlying cellular mechanisms by which steroids exert their therapeutic effects need to be fully elucidated. Here, we demonstrate that high-dose steroid treatment rapidly reduced the number of proinflammatory CXCR3+CD4+ T cells in the kidney by combining high-dimensional single-cell and morphological analyses of kidney biopsies from patients with antineutrophil cytoplasmic antibody–associated (ANCA-associated) crescentic glomerulonephritis. Using an experimental model of crescentic glomerulonephritis, we show that the steroid-induced decrease in renal CD4+ T cells is a consequence of reduced T cell recruitment, which is associated with an ameliorated disease course. Mechanistic in vivo and in vitro studies revealed that steroids act directly on renal tissue cells, such as tubular epithelial cells, but not on T cells, which resulted in an abolished renal expression of CXCL9 and CXCL10 as well as in the prevention of CXCR3+CD4+ T cell recruitment to the inflamed kidneys. Thus, we identified the CXCL9/CXCL10-CXCR3 axis as a previously unrecognized cellular and molecular target of glucocorticoids providing protection from immune-mediated pathology.
Jan-Hendrik Riedel, Lennart Robben, Hans-Joachim Paust, Yu Zhao, Nariaki Asada, Ning Song, Anett Peters, Anna Kaffke, Alina Borchers, Gisa Tiegs, Larissa Seifert, Nicola M. Tomas, Elion Hoxha, Ulrich O. Wenzel, Tobias B. Huber, Thorsten Wiech, Jan-Eric Turner, Christian F. Krebs, Ulf Panzer
Antibiotic-induced shifts in the indigenous gut microbiota influence normal skeletal maturation. Current theory implies that gut microbiota actions on bone occur through a direct gut/bone signaling axis. However, our prior work supports that a gut/liver signaling axis contributes to gut microbiota effects on bone. Our purpose was to investigate the effects of minocycline, a systemic antibiotic treatment for adolescent acne, on pubertal/postpubertal skeletal maturation. Sex-matched specific pathogen–free (SPF) and germ-free (GF) C57BL/6T mice were administered a clinically relevant minocycline dose from age 6–12 weeks. Minocycline caused dysbiotic shifts in the gut bacteriome and impaired skeletal maturation in SPF mice but did not alter the skeletal phenotype in GF mice. Minocycline administration in SPF mice disrupted the intestinal farnesoid X receptor/fibroblast growth factor 15 axis, a gut/liver endocrine axis supporting systemic bile acid homeostasis. Minocycline-treated SPF mice had increased serum conjugated bile acids that were farnesoid X receptor (FXR) antagonists, suppressed osteoblast function, decreased bone mass, and impaired bone microarchitecture and fracture resistance. Stimulating osteoblasts with the serum bile acid profile from minocycline-treated SPF mice recapitulated the suppressed osteogenic phenotype found in vivo, which was mediated through attenuated FXR signaling. This work introduces bile acids as a potentially novel mediator of gut/liver signaling actions contributing to gut microbiota effects on bone.
Matthew D. Carson, Amy J. Warner, Jessica D. Hathaway-Schrader, Vincenza L. Geiser, Joseph Kim, Joy E. Gerasco, William D. Hill, John J. Lemasters, Alexander V. Alekseyenko, Yongren Wu, Hai Yao, J. Ignacio Aguirre, Caroline Westwater, Chad M. Novince
Diamond-Blackfan anemia (DBA) is a genetic blood disease caused by heterozygous loss-of-function mutations in ribosomal protein (RP) genes, most commonly RPS19. The signature feature of DBA is hypoplastic anemia occurring in infants, although some older patients develop multilineage cytopenias with bone marrow hypocellularity. The mechanism of anemia in DBA is not fully understood and even less is known about the pancytopenia that occurs later in life, in part because patient hematopoietic stem and progenitor cells (HSPCs) are difficult to obtain, and the current experimental models are suboptimal. We modeled DBA by editing healthy human donor CD34+ HSPCs with CRISPR/Cas9 to create RPS19 haploinsufficiency. In vitro differentiation revealed normal myelopoiesis and impaired erythropoiesis, as observed in DBA. After transplantation into immunodeficient mice, bone marrow repopulation by RPS19+/− HSPCs was profoundly reduced, indicating hematopoietic stem cell (HSC) impairment. The erythroid and HSC defects resulting from RPS19 haploinsufficiency were partially corrected by transduction with an RPS19-expressing lentiviral vector or by Cas9 disruption of TP53. Our results define a tractable, biologically relevant experimental model of DBA based on genome editing of primary human HSPCs and they identify an associated HSC defect that emulates the pan-hematopoietic defect of DBA.
Senthil Velan Bhoopalan, Jonathan S. Yen, Thiyagaraj Mayuranathan, Kalin D. Mayberry, Yu Yao, Maria Angeles Lillo Osuna, Yoonjeong Jang, Janaka S.S. Liyanage, Lionel Blanc, Steven R. Ellis, Marcin W. Wlodarski, Mitchell J. Weiss
Vascular smooth muscle cell (SMC) phenotypic switching is widely recognized as a key mechanism responsible for the pathogenesis of several aortic diseases, such as aortic aneurysm. Cellular communication network factor 2 (CCN2), often upregulated in human pathologies and animal disease models, exerts myriad context-dependent biological functions. However, current understanding of the role of SMC-CCN2 in SMC phenotypic switching and its function in the pathology of abdominal aortic aneurysm (AAA) is lacking. Here, we show that SMC-restricted CCN2 deficiency causes AAA in the infrarenal aorta of angiotensin II–infused (Ang II–infused) hypercholesterolemic mice at a similar anatomic location to human AAA. Notably, the resistance of naive C57BL/6 WT mice to Ang II–induced AAA formation is lost upon silencing of CCN2 in SMC. Furthermore, the pro-AAA phenotype of SMC-CCN2-KO mice is recapitulated in a different model that involves the application of elastase–β-aminopropionitrile. Mechanistically, our findings reveal that CCN2 intersects with TGF-β signaling and regulates SMC marker expression. Deficiency of CCN2 triggers SMC reprograming associated with alterations in Krüppel-like factor 4 and contractile marker expression, and this reprograming likely contributes to the development of AAA in mice. These results identify SMC-CCN2 as potentially a novel regulator of SMC phenotypic switching and AA biology.
Yu Wang, Xuesong Liu, Qian Xu, Wei Xu, Xianming Zhou, Zhiyong Lin
BACKGROUND At the onset of exercise, the speed at which phosphocreatine (PCr) decreases toward a new steady state (PCr on-kinetics) reflects the readiness to activate mitochondrial ATP synthesis, which is secondary to Acetyl-CoA availability in skeletal muscle. We hypothesized that PCr on-kinetics are slower in metabolically compromised and older individuals and are associated with low carnitine acetyltransferase (CrAT) protein activity and compromised physical function.METHODS We applied 31P-magnetic resonance spectroscopy (31P-MRS) to assess PCr on-kinetics in 2 cohorts of volunteers. Cohort 1 included patients who had type 2 diabetes, were obese, were lean trained (VO2max > 55 mL/kg/min), and were lean untrained (VO2max < 45 mL/kg/min). Cohort 2 included young (20–30 years) and older (65–80 years) individuals with normal physical activity and older, trained individuals. Previous results of CrAT protein activity and acetylcarnitine content in muscle tissue were used to explore the underlying mechanisms of PCr on-kinetics, along with various markers of physical function.RESULTS PCr on-kinetics were significantly slower in metabolically compromised and older individuals (indicating mitochondrial inertia) as compared with young and older trained volunteers, regardless of in vivo skeletal muscle oxidative capacity (P < 0.001). Mitochondrial inertia correlated with reduced CrAT protein activity, low acetylcarnitine content, and functional outcomes (P < 0.001).CONCLUSION PCr on-kinetics are significantly slower in metabolically compromised and older individuals with normal physical activity compared with young and older trained individuals, regardless of in vivo skeletal muscle oxidative capacity, indicating greater mitochondrial inertia. Thus, PCr on-kinetics are a currently unexplored signature of skeletal muscle mitochondrial metabolism, tightly linked to functional outcomes. Skeletal muscle mitochondrial inertia might emerge as a target of intervention to improve physical function.TRIAL REGISTRATION NCT01298375 and NCT03666013 (clinicaltrials.gov).FUNDING RM and MH received an EFSD/Lilly grant from the European Foundation for the Study of Diabetes (EFSD). VS was supported by an ERC starting grant (grant 759161) “MRS in Diabetes.”
Rodrigo F. Mancilla, Lucas Lindeboom, Lotte Grevendonk, Joris Hoeks, Tim R. Koves, Deborah M. Muoio, Patrick Schrauwen, Vera Schrauwen-Hinderling, Matthijs K.C. Hesselink
The liver is a highly regenerative organ, yet the presence of a dedicated stem cell population remains controversial. Here, we interrogate a severe hepatocyte injury model in adult zebrafish to define that regeneration involves a stem cell population. After near-total hepatocyte ablation, single-cell transcriptomic and high-resolution imaging analyses throughout the entire regenerative timeline reveal that biliary epithelial cells undergo transcriptional and morphological changes to become hepatocytes. As a population, biliary epithelial cells give rise to both hepatocytes and biliary epithelial cells. Biliary epithelial cells proliferate and dedifferentiate to express hepatoblast transcription factors prior to hepatocyte differentiation. This process is characterized by increased MAPK, PI3K, and mTOR signaling, and chemical inhibition of these pathways impairs biliary epithelial cell proliferation and fate conversion. We conclude that, upon severe hepatocyte ablation in the adult liver, biliary epithelial cells act as facultative liver stem cells in an EGFR-PI3K-mTOR–dependent manner.
Isaac M. Oderberg, Wolfram Goessling
Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.
Qian Li, Kadambari Vijaykumar, Scott E. Phillips, Shah S. Hussain, Nha V. Huynh, Courtney M. Fernandez-Petty, Jacelyn E. Peabody Lever, Jeremy B. Foote, Janna Ren, Javier Campos-Gómez, Farah Abou Daya, Nathaniel W. Hubbs, Harrison Kim, Ezinwanne Onuoha, Evan R. Boitet, Lianwu Fu, Hui Min Leung, Linhui Yu, Thomas W. Detchemendy, Levi T. Schaefers, Jennifer L. Tipper, Lloyd J. Edwards, Sixto M. Leal Jr., Kevin S. Harrod, Guillermo J. Tearney, Steven M. Rowe
Podocyte injury and loss are key drivers of primary and secondary glomerular diseases, such as focal segmental glomerulosclerosis (FSGS) and diabetic kidney disease (DKD). We previously demonstrated the renoprotective role of protein S (PS) and its cognate tyrosine-protein kinase receptor, TYRO3, in models of FSGS and DKD and that their signaling exerts antiapoptotic and antiinflammatory effects to confer protection against podocyte loss. Among the 3 TAM receptors (TYRO3, AXL, and MER), only TYRO3 expression is largely restricted to podocytes, and glomerular TYRO3 mRNA expression negatively correlates with human glomerular disease progression. Therefore, we posited that the agonistic PS/TYRO3 signaling could serve as a potential therapeutic approach to attenuate glomerular disease progression. As PS function is not limited to TYRO3-mediated signal transduction but includes its anticoagulant activity, we focused on the development of TYRO3 agonists as an optimal therapeutic approach to glomerular disease. Among the small-molecule TYRO3 agonistic compounds screened, compound 10 (C-10) showed a selective activation of TYRO3 without any effects on AXL or MER. We also confirmed that C-10 directly binds to TYRO3, but not the other receptors. In vivo, C-10 attenuated proteinuria, glomerular injury, and podocyte loss in mouse models of Adriamycin-induced nephropathy and a db/db model of type 2 diabetes. Moreover, these renoprotective effects of C-10 were lost in Tyro3-knockout mice, indicating that C-10 is a selective agonist of TYRO3 activity that mitigates podocyte injury and glomerular disease. Therefore, C-10, a TYRO3 agonist, could be potentially developed as a new therapy for glomerular disease.
Fang Zhong, Hong Cai, Jia Fu, Zeguo Sun, Zhengzhe Li, David Bauman, Lois Wang, Bhaskar Das, Kyung Lee, John Cijiang He
Understanding persistence and evolution of B cell clones after COVID-19 infection and vaccination is crucial for predicting responses against emerging viral variants and optimizing vaccines. Here, we collected longitudinal samples from patients with severe COVID-19 every third to seventh day during hospitalization and every third month after recovery. We profiled their antigen-specific immune cell dynamics by combining single-cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq), and B cell receptor–Seq (BCR-Seq) with oligo-tagged antigen baits. While the proportion of Spike receptor binding domain–specific memory B cells (MBC) increased from 3 months after infection, the other Spike- and Nucleocapsid-specific B cells remained constant. All patients showed ongoing class switching and sustained affinity maturation of antigen-specific cells, and affinity maturation was not significantly increased early after vaccine. B cell analysis revealed a polyclonal response with limited clonal expansion; nevertheless, some clones detected during hospitalization, as plasmablasts, persisted for up to 1 year, as MBC. Monoclonal antibodies derived from persistent B cell families increased their binding and neutralization breadth and started recognizing viral variants by 3 months after infection. Overall, our findings provide important insights into the clonal evolution and dynamics of antigen-specific B cell responses in longitudinally sampled patients infected with COVID-19.
Lydia Scharf, Hannes Axelsson, Aikaterini Emmanouilidi, Nimitha R. Mathew, Daniel J. Sheward, Susannah Leach, Pauline Isakson, Ilya V. Smirnov, Emelie Marklund, Nicolae Miron, Lars-Magnus Andersson, Magnus Gisslén, Ben Murrell, Anna Lundgren, Mats Bemark, Davide Angeletti