The use of patient-derived organoids (PDOs) to characterize therapeutic sensitivity and resistance is a promising precision medicine approach, and its potential to inform clinical decisions is now being tested in several large multi-institutional clinical trials. PDOs are cultivated in extracellular matrix from basement membrane extracts (BMEs) that are most commonly acquired commercially. Each clinical site utilizes distinct BME lots and may be restricted due to the availability of commercial BME sources. However, the impact of different sources of BMEs on organoid drug response is unknown. Here, we tested the impact of BME source on proliferation, drug response, and gene expression in mouse and human pancreatic ductal adenocarcinoma (PDA) organoids. Both human and mouse organoids displayed increased proliferation in Matrigel (Corning) compared to Cultrex (RnD) and UltiMatrix (RnD). However, we observed no substantial impact on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources, and PDOs maintained their Classical or Basal-like designation. Overall, we find that BME source (Matrigel, Cultrex, UltiMatrix) does not shift PDO dose-response curves and drug testing results, indicating that PDO pharmacotyping is a robust approach for precision medicine.
Jan C. Lumibao, Shira R. Okhovat, Kristina L. Peck, Xiaoxue Lin, Kathryn Lande, Shira Yomtoubian, Isabella Ng, Herve Tiriac, Andrew M. Lowy, Jingjing Zou, Dannielle D. Engle
There is great interest in identifying signaling pathways that promote cardiac repair after myocardial infarction (MI). Prior studies suggest a beneficial role for IL13 signaling in neonatal heart regeneration, however, the cell types mediating cardiac regeneration and the extent of IL13 signaling in the adult heart post-injury are unknown. We identified an abundant source of IL13 and the related cytokine, IL4, in neonatal cardiac type 2 innate lymphoid cells (ILC2s), however, ILC2 production of IL13 and IL4 as well as ILC2 frequency declined precipitously in adult hearts. In agreement with this finding, IL13 receptor deletion in macrophages impaired cardiac function and delayed scar clearance after neonatal MI. By using a combination of recombinant IL13 (rIL13) administration and cell-specific IL13 receptor genetic deletion models we found that IL13 signaling specifically to macrophages significantly promotes cardiac functional recovery after MI in adult mice. Single cell RNA sequencing revealed a sub-population of macrophages appearing in the heart early after injury only in response to rIL13 administration. These IL13 induced macrophages are independent of classically defined alternatively activated macrophages, are highly efferocytotic and can be identified in vivo by expression of IL1R2. IL1R2+ macrophages are induced upon rIL13 administration in adult mice and depend on IL13 signaling directly to macrophages. Collectively, we elucidate a strongly pro-reparative role for IL13 signaling directly to macrophages following cardiac injury. While this pathway is active in pro-regenerative neonatal stages, re-activation of macrophage IL13 signaling is required to promote cardiac functional recovery in adults.
Santiago Alvarez-Argote, Samantha J. Paddock, Michael A. Flinn, Caelan W. Moreno, Makenna C. Knas, Victor A. Almeida, Sydney L. Buday, Amirala Bakhshian Nik, Michaela Patterson, Yi-Guang Chen, Chien-Wei Lin, Caitlin C. O'Meara
Cachexia is a debilitating skeletal muscle wasting condition for which we currently lack effective treatments. In the context of cancer, certain chemotherapeutics cause DNA damage and cellular senescence. Senescent cells exhibit chronic activation of the transcription factor nuclear factor (NF)-κB, a known mediator of the pro-inflammatory senescence-associated secretory phenotype (SASP) and skeletal muscle atrophy. Thus, targeting NF-κB represents a logical therapeutic strategy to alleviate unintended consequences of genotoxic drugs. Herein, we show that treatment with the IKK/NF-κB inhibitor SR12343 during a course of chemotherapy reduces markers of cellular senescence and the SASP in liver, skeletal muscle, and circulation and, correspondingly, attenuates features of skeletal muscle pathology. Lastly, we demonstrate SR12343 mitigates chemotherapy-induced reductions in body weight, lean mass, fat mass, and muscle strength. These findings support senescent cells as a promising druggable target to counteract the SASP and skeletal muscle wasting in the context of chemotherapy.
Davis A. Englund, Alyssa M. Jolliffe, Gabriel J. Hanson, Zaira Aversa, Xu Zhang, Xinyi Jiang, Thomas A. White, Lei Zhang, David G. Monroe, Paul D. Robbins, Laura J. Niedernhofer, Theodore M. Kamenecka, Sundeep Khosla, Nathan K. LeBrasseur
The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP–indian hedgehog (Ihh) feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and trace the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with Patched-1 (Ptch1) floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large concentric clonally expanded cell populations within the resting zone (‘patched roses’) and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell-descendants migrated away from the growth plate and eventually transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.
Shion Orikasa, Yuki Matsushita, Hiroaki Manabe, Michael Fogge, Zachary J. Lee, Koji Mizuhashi, Naoko Sakagami, Wanida Ono, Noriaki Ono
Hyperuricemia is implicated in numerous pathologies but the mechanisms underlying uric acid production are poorly understood. Using a combination of mouse studies, cultured cell studies, and human serum samples, we sought to determine the cellular source of uric acid. In mice, fasting and glucocorticoid treatment increased serum uric acid and uric acid release from ex vivo incubated skeletal muscle. In vitro, glucocorticoids and the transcription factor FoxO3 increased purine nucleotide degradation and purine release from differentiated muscle cells, which coincided with the transcriptional upregulation of AMP deaminase 3, a rate-limiting enzyme in adenine nucleotide degradation. Heavy isotope tracing during co-culture experiments revealed that oxidation of muscle purines to uric acid required their transfer from muscle cells to a cell type that expresses xanthine oxidoreductase, such as endothelial cells. Lastly, in healthy women, matched for age and body composition, serum uric acid was greater in individuals scoring below average on standard physical function assessments. Together, these studies reveal skeletal muscle purine degradation is an underlying driver of uric acid production, with the final step of uric acid production occurring primarily in a non-muscle cell type. This suggests that skeletal muscle fiber purine degradation may represent a therapeutic target to reduce serum uric acid and treat numerous pathologies.
Spencer G. Miller, Catalina Matias, Paul S. Hafen, Andrew S. Law, Carol A. Witczak, Jeffrey J. Brault
Circadian rhythm dysfunction is a hallmark of Parkinson Disease (PD), and diminished expression of the core clock gene Bmal1 has been described in PD patients. BMAL1 is required for core circadian clock function, but also serves non-rhythmic functions. Germline Bmal1 deletion can cause brain oxidative stress and synapse loss in mice, and can exacerbate dopaminergic neurodegeneration in response to the toxin MPTP. Here we examined the impact of cell type-specific Bmal1 deletion on dopaminergic neuron viability in vivo. We observed that global, post-natal deletion of Bmal1 caused spontaneous loss of tyrosine hydroxylase-positive (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc). This was not replicated by light-induced disruption of behavioral circadian rhythms, and was not induced by astrocyte- or microglia-specific Bmal1 deletion. However, either pan-neuronal or TH neuron-specific Bmal1 deletion caused cell-autonomous loss of TH+ neurons in the SNpc. Bmal1 deletion did not change the percentage of TH neuron loss after alpha-synuclein fibril injection, though Bmal1 KO mice had fewer TH neurons at baseline. Transcriptomic analysis revealed dysregulation of pathways involved in oxidative phosphorylation and Parkinson Disease. These findings demonstrate a cell-autonomous role for BMAL1 in regulating dopaminergic neuronal survival, and may have important implications for neuroprotection in PD.
Michael K. Kanan, Patrick W. Sheehan, Jessica N. Haines, Pedro G. Gomez, Adya Dhuler, Collin J. Nadarajah, Zachary M. Wargel, Brittany M. Freeberg, Hemanth R. Nelvagal, Mariko Izumo, Joseph S. Takahashi, Jonathan D. Cooper, Albert A. Davis, Erik S. Musiek
Drug-induced liver injury (DILI), especially acetaminophen overdose, is the leading cause of acute liver failure. Pregnane X receptor (PXR) is a nuclear receptor and the master regulator of drug metabolism. Aberrant activation of PXR plays a pathogenic role in the acetaminophen hepatotoxicity. Here, we aimed to examine the PXR S-nitrosylation (SNO) in response to acetaminophen. We found that PXR was S-nitrosylated in hepatocytes and the mouse livers after exposure to acetaminophen or S-nitrosoglutathione (GSNO). Mass-spectrometry and site-directed mutagenesis identified the cysteine 307 as the primary residue for SNO-modification. In hepatocytes, SNO suppressed both agonist (rifampicin and SR12813)-induced and constitutively active PXR (VP-PXR) activations. Furthermore, in acetaminophen overdosed mouse livers, PXR protein was decreased at the centrilobular regions overlapping with increased SNO. In PXR-deficient (PXR-/-) mice, replenishing the livers with the SNO-deficient PXR significantly aggravated hepatic necrosis, increased HMGB1 release, and exacerbated liver injury and inflammation. Particularly, we demonstrated that S-nitrosoglutathione reductase (GSNOR) inhibitor N6022 promoted hepatoprotection by increasing the levels of PXR S-nitrosylation. In conclusion, PXR is post-translationally modified by S-nitrosylation in hepatocytes in response to acetaminophen. This modification mitigated the acetaminophen-induced PXR hyperactivity. It may serve as a target for therapeutical intervention.
Qi Cui, Tingting Jiang, Xinya Xie, Haodong Wang, Lei Qian, Yanyan Cheng, Qiang Li, Tingxu Lu, Qinyu Yao, Jia Liu, Baochang Lai, Chang Chen, Lei Xiao, Nanping Wang
Tuberculosis, a chronic infectious disease caused by a single pathogen, holds the highest mortality rate worldwide. RNA-binding proteins (RBPs) are involved in autophagy — a key defense mechanism against Mycobacterium tuberculosis (Mtb) infection — by modulating RNA stability and forming intricate regulatory networks. However, the functions of host RBPs during Mtb infection remain relatively unexplored. ZNFX1, a conserved RBP critically involved in immune deficiency diseases and mycobacterial infections, is significantly upregulated in Mtb-infected macrophages. Here, we aimed to explore the immune regulatory functions of ZNFX1 during Mtb infection. We observed that Znfx1 knockout markedly compromised the multifaceted immune responses mediated by macrophages. This compromise resulted in reduced phagocytosis, suppressed macrophage activation, increased Mtb burden, progressive lung tissue injury, and chronic inflammation in Mtb-infected mice. Mechanistic investigations revealed that the absence of ZNFX1 inhibited autophagy, consequently mediating immune suppression. ZNFX1 critically maintained AMPK-regulated autophagic flux by stabilizing Prkaa2 mRNA, which encodes a key catalytic α subunit of AMPK, through its zinc finger region. This process contributed to Mtb growth suppression. These findings reveal a function of ZNFX1 in establishing anti-Mtb immune responses, enhancing our understanding of the roles of RBPs in tuberculosis immunity and providing a promising approach to bolster anti-tuberculosis immunotherapy.
Honglin Liu, Zhenyu Han, Liru Chen, Jing Zhang, Zhanqing Zhang, Yaoxin Chen, Feichang Liu, Ke Wang, Jieyu Liu, Na Sai, Xinying Zhou, Chaoying Zhou, Shengfeng Hu, Qian Wen, Li Ma
Pulmonary fibrosis is a chronic and often fatal disease. The pathogenesis is characterized by aberrant repair of lung parenchyma resulting in loss of physiological homeostasis, respiratory failure and death. The immune response in pulmonary fibrosis is dysregulated. The gut microbiome is a key regulator of immunity. The role of the gut microbiome in regulating the pulmonary immunity in lung fibrosis is poorly understood. Here, we determine the impact of gut microbiota on pulmonary fibrosis in C57BL/6 mice derived from different vendors (C57BL/6J and C57BL/6NCrl). We use germ free models, fecal microbiota transplantation and cohousing to transmit gut microbiota. Metagenomic studies of feces establish keystone species between sub-strains. Pulmonary fibrosis is microbiota dependent in C57BL/6 mice. Gut microbiota are distinct by β diversity (PERMANOVA P<0.001) and α diversity (P<0.0001). Mortality and lung fibrosis are attenuated in C57BL/6NCrl mice. Elevated CD4+ IL-10+ T cells and lower IL-6 occur in C57BL/6NCrl mice. Horizontal transmission of microbiota by cohousing attenuates mortality in C57BL/6J mice and promotes a transcriptionally altered pulmonary immunity. Temporal changes in lung and gut microbiota demonstrates that gut microbiota contribute largely to immunological phenotype. Key regulatory gut microbiota contribute to lung fibrosis generating rationale for human studies.
Stephen J. Gurczynski, Jay H. Lipinski, Joshua Y. Strauss, Shafiul Alam, Gary B. Huffnagle, Piyush Ranjan, Lucy H. Kennedy, Bethany B. Moore, David N. O'Dwyer
Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-seq comparing osteoblasts differentiated from Ezh2R684C/+ and Ezh2+/+ bone marrow mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A/6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state, and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.
Christine W. Gao, WanYing Lin, Ryan C. Riddle, Priyanka Kushwaha, Leandros Boukas, Hans T. Björnsson, Kasper D. Hansen, Jill A. Fahrner
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