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
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
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
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
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
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
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
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
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
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
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