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 perform head-to-head comparisons between the transcriptomes of fresh primary (1o) adult human AEC2s, their cultured progeny, and human induced pluripotent stem cell-derived AEC2s (iAEC2s). We find each population occupies a distinct transcriptomic space with cultured AEC2s (1o and iAEC2s) exhibiting similarities to and differences from freshly purified 1o cells. Across each cell type, we find an inverse relationship between proliferative and maturation states, with pre-culture 1o AEC2s being most quiescent/mature and iAEC2s being most proliferative/least mature. Cultures of either type of human AEC2 do not generate detectable alveolar type 1 cells in these defined conditions; however, a subset of iAEC2s co-cultured with fibroblasts acquires 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 1o and engineered AEC2s, two 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
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 severe COVID-19 patients every third to seventh day during hospitalization and every third month after recovery. We profiled the antigen-specific immune cell dynamics by combining single cell RNA-Seq, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-Seq, B cell receptor (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, which 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 one 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 COVID-19 infected patients.
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
FOXD1+ 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 deletion 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
Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability exceeding genic effects. Dysregulated metabolism and immune cell function are key disease modifiers. The tryptophan metabolites, kynurenines, produced through 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 wildtype. Further, IDO1 levels were increased in ADPKD cell lines. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity as measured by %kidney weight/body weight and cystic index. 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 similar changes in the CME as 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 novel 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
Mitochondria are dynamic organelles responsible for energy production and many processes central to cellular function. Alterations in mitochondrial function is associated with human fibrotic lung diseases, including idiopathic pulmonary fibrosis (IPF). Pulmonary fibrosis is characterized by stiffening of the extracellular matrix (ECM). 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 identified mitochondria as an important mechanotransducer at the intersection between extracellular mechanical signals and durotactic lung fibroblast migration. 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 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. Our 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 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 IPF.
Ting Guo, Chun-sun Jiang, Shan-Zhong Yang, Yi Zhu, Chao He, A. Brent Carter, Veena B. Antony, Hong Peng, Yong Zhou
Although glycogen synthase kinase β (Gsk3β) has been shown to regulate tissue inflammation, whether and how it regulates inflammation resolution vs. 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, but also facilitated the restoration of liver homeostasis. Depletion of Kupffer cells (KCs) 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 the Gsk3β KO mice. In CD11b-DTR mice, Gsk3β deficient bone marrow-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 pro-resolving function of liver infiltrating macrophages in 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
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. Purpose was to investigate the effects of minocycline, a systemic antibiotic treatment for adolescent acne, on pubertal/post-pubertal 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(FXR)-fibroblast growth factor 15(FGF15) axis, a gut-liver endocrine axis supporting systemic bile acid homeostasis. Minocycline-treated SPF mice had increased serum conjugated bile acids that are FXR antagonists, suppressed osteoblast function, decreased bone mass, 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 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 D. Kim, Joy E. Gerasco, William D. Hill, John J. Lemasters, Alexander V. Alekseyenko, Yongren Wu, Hai Yao, Jose I. Aguirre, Caroline Westwater, Chad M. Novince
Metastatic clear cell renal cell carcinomas (ccRCC) are resistant to DNA damaging chemotherapies, limiting therapeutic options for patients whose tumours are resistant to tyrosine kinase inhibitors and/or immune checkpoint therapies. Here we show that mouse and human ccRCC are frequently characterised by high levels of endogenous DNA damage and that cultured ccRCC cells exhibit intact cellular responses to chemotherapy-induced DNA damage. We identify that pharmacological inhibition of the DNA damage sensing kinase ATR with the orally administered, potent and selective drug M4344 (also called gartisertib) induces anti-proliferative effects in ccRCC cells due to replication stress and the accumulation of DNA damage in S phase. In some cells, DNA damage persists into subsequent G2/M and G1 phases, leading to the frequent accumulation of micronuclei. Daily single agent treatment with M4344 inhibited the growth of ccRCC xenograft tumours. M4344 synergises with chemotherapeutic drugs including cisplatin and carboplatin and the PARP inhibitor olaparib in mouse and human ccRCC cells. Weekly M4344 plus cisplatin treatment showed in vivo therapeutic synergy in ccRCC xenografts and was efficacious in an autochthonous mouse ccRCC model. These studies identify ATR inhibition as a potential novel therapeutic option for ccRCC.
Philipp Seidel, Anne Rubarth, Kyra Zodel, Asin Peighambari, Felix Neumann, Yannick Federkiel, Hsin Huang, Rouven Hoefflin, Mojca Adlesic, Christian Witt, David J. Hoffmann, Patrick Metzger, Ralph K. Lindemann, Frank T. Zenke, Christoph Schell, Melanie Boerries, Dominik von Elverfeldt, Wilfried Reichardt, Marie Follo, Joachim Albers, Ian J. Frew
Carbohydrate Responsive 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 (HGF). We demonstrate that HGFAC KO mice have 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 enhances lipid and glucose homeostasis, which may be mediated in part through actions to activate hepatic PPARγ activity. Together, our studies show that ChREBP mediates an adaptive response to overnutrition via activation of HGFAC in the liver to preserve glucose and lipid homeostasis.
Ashot Sargsyan, Ludivine Doridot, Sarah Anissa 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.-Y. Tsai, Mark A. Herman
Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulations play a critical role. Lung tumor suppressor 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 induces GPRC5A repression both in vitro and in vivo. Intriguingly, trans-activation of NF-κB downstream targets is not required, but the trans-activation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in GPRC5A promoter. Instead, p65 was complexed with RARα/β, and recruited to the RA-response element (RARE) site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complex, and suppressed transcription. Noticeably, phosphorylation on Serine276 of p65 is required for interaction with RARα/β and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression is through suppression of histone H3K9ac, but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a HDAC inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via 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, Yugene Chin, Qi Wang, Binhua P. Zhou, Jiong Deng
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