In this issue, Kashgari et al. report that the epidermal transcription factor Grainyhead like-3 (GRHL3) controls keratinocyte migration during wound healing via FSCN1 expression. The cover image is modified from a transmission electron micrograph that shows two migrating keratinocytes at the wound front in mouse skin tissue.
Hyperstimulation of the cholecystokinin 1 receptor (CCK1R), a G protein–coupled receptor (GPCR), in pancreatic acinar cells is commonly used to induce pancreatitis in rodents. Human pancreatic acinar cells lack CCK1R but express cholinergic receptor muscarinic 3 (M3R), another GPCR. To test whether M3R activation is involved in pancreatitis, a mutant M3R was conditionally expressed in pancreatic acinar cells in mice. This mutant receptor loses responsiveness to its native ligand, acetylcholine, but can be activated by an inert small molecule, clozapine-N-oxide (CNO). Intracellular calcium and amylase were elicited by CNO in pancreatic acinar cells isolated from mutant M3R mice but not WT mice. Similarly, acute pancreatitis (AP) could be induced by a single injection of CNO in the transgenic mice but not WT mice. Compared with the cerulein-induced AP, CNO caused more widespread acinar cell death and inflammation. Furthermore, chronic pancreatitis developed at 4 weeks after 3 episodes of CNO-induced AP. In contrast, in mice with 3 recurrent episodes of cerulein-included AP, pancreas histology was restored in 4 weeks. Furthermore, the M3R antagonist ameliorated the severity of cerulein-induced AP in WT mice. We conclude that M3R activation can cause the pathogenesis of pancreatitis. This model may provide an alternative approach for pancreatitis research.
Jianhua Wan, Jiale Wang, Larry E. Wagner II, Oliver H. Wang, Fu Gui, Jiaxiang Chen, Xiaohui Zhu, Ashley N. Haddock, Brandy H. Edenfield, Brian Haight, Debabrata Mukhopadhyay, Ying Wang, David I. Yule, Yan Bi, Baoan Ji
Mitochondrial dysfunction is a major pathophysiological contributor to the progression of Parkinson’s disease (PD); however, whether it contributes to epigenetic dysregulation remains unknown. Here, we show that both chemically and genetically driven mitochondrial dysfunctions share a common mechanism of epigenetic dysregulation. Under both scenarios, lysine 27 acetylation of likely variant H3.3 (H3.3K27ac) increased in dopaminergic neuronal models of PD, thereby opening that region to active enhancer activity via H3K27ac. These vulnerable epigenomic loci represent potential transcription factor motifs for PD pathogenesis. We further confirmed that mitochondrial dysfunction induces H3K27ac in ex vivo and in vivo (MitoPark) neurodegenerative models of PD. Notably, the significantly increased H3K27ac in postmortem PD brains highlights the clinical relevance to the human PD population. Our results reveal an exciting mitochondrial dysfunction-metabolism-H3K27ac-transcriptome axis for PD pathogenesis. Collectively, the mechanistic insights link mitochondrial dysfunction to epigenetic dysregulation in dopaminergic degeneration and offer potential new epigenetic intervention strategies for PD.
Minhong Huang, Dan Lou, Adhithiya Charli, Dehui Kong, Huajun Jin, Gary Zenitsky, Vellareddy Anantharam, Arthi Kanthasamy, Zhibin Wang, Anumantha G. Kanthasamy
The creatine transporter (CrT) maintains brain creatine (Cr) levels, but the effects of its deficiency on energetics adaptation under stress remain unclear. There are also no effective treatments for CrT deficiency, the second most common cause of X-linked intellectual disabilities. Herein, we examined the consequences of CrT deficiency in brain energetics and stress-adaptation responses plus the effects of intranasal Cr supplementation. We found that CrT-deficient (CrT–/y) mice harbored dendritic spine and synaptic dysgenesis. Nurtured newborn CrT–/y mice maintained baseline brain ATP levels, with a trend toward signaling imbalance between the p-AMPK/autophagy and mTOR pathways. Starvation elevated the signaling imbalance and reduced brain ATP levels in P3 CrT–/y mice. Similarly, CrT–/y neurons and P10 CrT–/y mice showed an imbalance between autophagy and mTOR signaling pathways and greater susceptibility to cerebral hypoxia-ischemia and ischemic insults. Notably, intranasal administration of Cr after cerebral ischemia increased the brain Cr/N-acetylaspartate ratio, partially averted the signaling imbalance, and reduced infarct size more potently than intraperitoneal Cr injection. These findings suggest important functions for CrT and Cr in preserving the homeostasis of brain energetics in stress conditions. Moreover, intranasal Cr supplementation may be an effective treatment for congenital CrT deficiency and acute brain injury.
Hong-Ru Chen, Xiaohui Zhang-Brotzge, Yury M. Morozov, Yuancheng Li, Siming Wang, Helen Heju Zhang, Irena S. Kuan, Elizabeth M. Fugate, Hui Mao, Yu-Yo Sun, Pasko Rakic, Diana M. Lindquist, Ton DeGrauw, Chia-Yi Kuan
The migrating keratinocyte wound front is required for skin wound closure. Despite significant advances in wound healing research, we do not fully understand the molecular mechanisms that orchestrate collective keratinocyte migration. Here, we show that, in the wound front, the epidermal transcription factor Grainyhead like-3 (GRHL3) mediates decreased expression of the adherens junction protein E-cadherin; this results in relaxed adhesions between suprabasal keratinocytes, thus promoting collective cell migration and wound closure. Wound fronts from mice lacking GRHL3 in epithelial cells (Grhl3-cKO) have lower expression of Fascin-1 (FSCN1), a known negative regulator of E-cadherin. Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) on wounded keratinocytes shows decreased wound-induced chromatin accessibility near the Fscn1 gene in Grhl3-cKO mice, a region enriched for GRHL3 motifs. These data reveal a wound-induced GRHL3/FSCN1/E-cadherin pathway that regulates keratinocyte-keratinocyte adhesion during wound-front migration; this pathway is activated in acute human wounds and is altered in diabetic wounds in mice, suggesting translational relevance.
Ghaidaa Kashgari, Sanan Venkatesh, Samuel Refuerzo, Brandon Pham, Anita Bayat, Rachel Herndon Klein, Raul Ramos, Albert Paul Ta, Maksim V. Plikus, Ping H. Wang, Bogi Andersen
Stromal interaction molecule 1 (STIM1), the sarcoplasmic reticulum (SR) transmembrane protein, activates store-operated Ca2+ entry (SOCE) in skeletal muscle and, thereby, coordinates Ca2+ homeostasis, Ca2+-dependent gene expression, and contractility. STIM1 occupies space in the junctional SR membrane of the triads and the longitudinal SR at the Z-line. How STIM1 is organized and is retained in these specific subdomains of the SR is unclear. Here, we identified desmin, the major type III intermediate filament protein in muscle, as a binding partner for STIM1 based on a yeast 2-hybrid screen. Validation of the desmin-STIM1 interaction by immunoprecipitation and immunolocalization confirmed that the CC1-SOAR domains of STIM1 interact with desmin to enhance STIM1 oligomerization yet limit SOCE. Based on our studies of desmin-KO mice, we developed a model wherein desmin connected STIM1 at the Z-line in order to regulate the efficiency of Ca2+ refilling of the SR. Taken together, these studies showed that desmin-STIM1 assembles a cytoskeletal-SR connection that is important for Ca2+ signaling in skeletal muscle.
Hengtao Zhang, Victoria Graham Bryson, Chaojian Wang, TianYu Li, Jaclyn P. Kerr, Rebecca Wilson, Deborah M. Muoio, Robert J. Bloch, Christopher Ward, Paul B. Rosenberg
Chronic obstructive pulmonary disease (COPD) is mainly caused by cigarette smoking and characterized by chronic inflammation in vulnerable individuals. However, it is unknown how genetic factors may shape chronic inflammation in COPD. To understand how hedgehog interacting protein, encoded by HHIP gene identified in the genome-wide association study in COPD, plays a role in inflammation, we utilized Hhip+/– mice that present persistent inflammation and emphysema upon aging similar to that observed in human COPD. By performing single-cell RNA sequencing of the whole lung from mice at different ages, we found that Hhip+/– mice developed a cytotoxic immune response with a specific increase in killer cell lectin-like receptor G1–positive CD8+ T cells with upregulated Ifnγ expression recapitulating human COPD. Hhip expression was restricted to a lung fibroblast subpopulation that had increased interaction with CD8+ T lymphocytes in Hhip+/– compared with Hhip+/+ during aging. Hhip-expressing lung fibroblasts had upregulated IL-18 pathway genes in Hhip+/– lung fibroblasts, which was sufficient to drive increased levels of IFN-γ in CD8+ T cells ex vivo. Our finding provides insight into how a common genetic variation contributes to the amplified lymphocytic inflammation in COPD.
Jeong H. Yun, ChangHee Lee, Tao Liu, Siqi Liu, Edy Y. Kim, Shuang Xu, Jeffrey L. Curtis, Luca Pinello, Russell P. Bowler, Edwin K. Silverman, Craig P. Hersh, Xiaobo Zhou
Xanthine oxidoreductase (XOR) is an enzyme that catalyzes hypoxanthine to xanthine and xanthine to uric acid, respectively. However, the underlying mechanisms of increased plasma XOR and its pathological roles in systemic diseases, such as atherosclerosis, are not fully understood. In this study, we found that changes in plasma XOR activity after bariatric surgery closely associated with those in liver enzymes, but not with those in BMI. In a mouse model of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH), plasma XOR activity markedly increased. Besides, purine catabolism was accelerated in the plasma per se of NASH mice and human patients with high XOR activity. In our NASH mice, we observed an increased vascular neointima formation consisting of dedifferentiated vascular smooth muscle cells (SMCs), which was significantly attenuated by topiroxostat, a selective XOR inhibitor. In vitro, human liver S9–derived XOR promoted proliferation of SMCs with phenotypic modulation and induced ROS production by catabolizing hypoxanthine released from human endothelial cells. Collectively, the results from human and mouse models suggest that increased plasma XOR activity, mainly explained by excess hepatic leakage, was involved in the pathogenesis of vascular injury, especially in NAFLD/NASH conditions.
Yusuke Kawachi, Yuya Fujishima, Hitoshi Nishizawa, Takashi Nakamura, Seigo Akari, Takayo Murase, Takuro Saito, Yasuhiro Miyazaki, Hirofumi Nagao, Shiro Fukuda, Shunbun Kita, Naoto Katakami, Yuichiro Doki, Norikazu Maeda, Iichiro Shimomura
Type 2 DCs (DC2s) comprise the majority of conventional DCs within most tumors; however, little is known about their ability to initiate and sustain antitumor immunity, as most studies have focused on antigen cross-presenting DC1s. Here, we report that DC2 infiltration identified by analysis of multiple human cancer data sets showed a significant correlation with survival across multiple human cancers, with the benefit being seen in tumors resistant to cytotoxic T cell control. Characterization of DC subtype infiltration into an immunotherapy-resistant model of breast cancer revealed that impairment of DC1s through 2 unique models resulted in enhanced DC2 functionality and improved tumor control. BATF3 deficiency depleted intratumoral DC1s, which led to increased DC2 lymph node migration and CD4+ T cell activation. Enhancing DC2 stimulatory potential by genetic deletion of Hsp90b1 (encoding molecular chaperon GP96) led to a similar enhancement of T cell immunity and improved survival in a spontaneous breast cancer model. These data highlight the therapeutic and prognostic potential of DC2s within checkpoint blockade–resistant tumors.
Stephen Iwanowycz, Soo Ngoi, Yingqi Li, Megan Hill, Christopher Koivisto, Melodie Parrish, Beichu Guo, Zihai Li, Bei Liu
Pancreatic ductal adenocarcinoma (PDAC) tumors are characterized by a desmoplastic reaction resulting in dense deposition of collagen that is known to promote cancer progression. A central mediator of protumorigenic collagen signaling is the receptor tyrosine kinase discoid domain receptor 1 (DDR1). DDR1 is a critical driver of a mesenchymal and invasive cancer cell PDAC phenotype. Previous studies have demonstrated that genetic or pharmacologic inhibition of DDR1 reduces PDAC tumorigenesis and metastasis. Here, we investigated whether DDR1 signaling has cancer cell nonautonomous effects that promote PDAC progression and metastasis. We demonstrate that collagen-induced DDR1 activation in cancer cells is a major stimulus for CXCL5 production, resulting in the recruitment of tumor-associated neutrophils (TANs), the formation of neutrophil extracellular traps (NETs), and subsequent cancer cell invasion and metastasis. Moreover, we have identified that collagen-induced CXCL5 production was mediated by a DDR1/PKCθ/SYK/NF-κB signaling cascade. Together, these results highlight the critical contribution of the collagen I–DDR1 interaction in the formation of an immune microenvironment that promotes PDAC metastasis.
Jenying Deng, Yaan Kang, Chien-Chia Cheng, Xinqun Li, Bingbing Dai, Matthew H. Katz, Taoyan Men, Michael P. Kim, Eugene A. Koay, Huocong Huang, Rolf A. Brekken, Jason B. Fleming
Development of primary liver cancer is a multistage process. Detailed understanding of sequential epigenetic alterations is largely missing. Here, we performed Infinium Human Methylation 450k BeadChips and RNA-Seq analyses for genome-wide methylome and transcriptome profiling of cirrhotic liver (n = 7), low- (n = 4) and high-grade (n = 9) dysplastic lesions, and early (n = 5) and progressed (n = 3) hepatocellular carcinomas (HCC) synchronously detected in 8 patients with HCC with chronic hepatitis B infection. Integrative analyses of epigenetically driven molecular changes were identified and validated in 2 independent cohorts comprising 887 HCCs. Mitochondrial DNA sequencing was further employed for clonality analyses, indicating multiclonal origin in the majority of investigated HCCs. Alterations in DNA methylation progressively increased from liver cirrhosis (CL) to dysplastic lesions and reached a maximum in early HCCs. Associated early alterations identified by Ingenuity Pathway Analysis (IPA) involved apoptosis, immune regulation, and stemness pathways, while late changes centered on cell survival, proliferation, and invasion. We further validated 23 putative epidrivers with concomitant expression changes and associated with overall survival. Functionally, Striatin 4 (STRN4) was demonstrated to be epigenetically regulated, and inhibition of STRN4 significantly suppressed tumorigenicity of HCC cell lines. Overall, application of integrative genomic analyses defines epigenetic driver alterations and provides promising targets for potentially novel therapeutic approaches.
Carolin Czauderna, Alicia Poplawski, Colm J. O’Rourke, Darko Castven, Benjamín Pérez-Aguilar, Diana Becker, Stephanie Heilmann-Heimbach, Margarete Odenthal, Wafa Amer, Marcel Schmiel, Uta Drebber, Harald Binder, Dirk A. Ridder, Mario Schindeldecker, Beate K. Straub, Peter R. Galle, Jesper B. Andersen, Snorri S. Thorgeirsson, Young Nyun Park, Jens U. Marquardt
BACKGROUND Naturally acquired immunity to malaria is incompletely understood. We used controlled human malaria infection (CHMI) to study the impact of past exposure on malaria in Kenyan adults in relation to infection with a non-Kenyan parasite strain.METHODS We administered 3.2 × 103 aseptic, purified, cryopreserved Plasmodium falciparum sporozoites (Sanaria PfSPZ Challenge, NF54 West African strain) by direct venous inoculation and undertook clinical monitoring and serial quantitative PCR (qPCR) of the 18S ribosomal RNA gene. The study endpoint was met when parasitemia reached 500 or more parasites per μL blood, clinically important symptoms were seen, or at 21 days after inoculation. All volunteers received antimalarial drug treatment upon meeting the endpoint.RESULTS One hundred and sixty-one volunteers underwent CHMI between August 4, 2016, and February 14, 2018. CHMI was well tolerated, with no severe or serious adverse events. Nineteen volunteers (11.8%) were excluded from the analysis based on detection of antimalarial drugs above the minimal inhibitory concentration or parasites genotyped as non-NF54. Of the 142 volunteers who were eligible for analysis, 26 (18.3%) had febrile symptoms and were treated; 30 (21.1%) reached 500 or more parasites per μL and were treated; 53 (37.3%) had parasitemia without meeting thresholds for treatment; and 33 (23.2%) remained qPCR negative.CONCLUSION We found that past exposure to malaria, as evidenced by location of residence, in some Kenyan adults can completely suppress in vivo growth of a parasite strain originating from outside Kenya.TRIAL REGISTRATION ClinicalTrials.gov NCT02739763.FUNDING Wellcome Trust.
Melissa C. Kapulu, Patricia Njuguna, Mainga Hamaluba, Domtila Kimani, Joyce M. Ngoi, Janet Musembi, Omar Ngoto, Edward Otieno, Peter F. Billingsley, the Controlled Human Malaria Infection in Semi-Immune Kenyan Adults (CHMI-SIKA) Study Team
The mechanism controlling long-chain fatty acid (LCFA) mobilization from adipose tissue is not well understood. Here, we investigated how the LCFA transporter CD36 regulates this process. By using tissue-specific KO mouse models, we showed that CD36 in adipocytes and endothelial cells mediated both LCFA deposition into and release from adipose tissue. We demonstrated the role of adipocytic and endothelial CD36 in promoting tumor growth and chemoresistance conferred by adipose tissue–derived LCFAs. We showed that dynamic cysteine S-acylation of CD36 in adipocytes, endothelial cells, and cancer cells mediated intercellular LCFA transport. We demonstrated that lipolysis induction in adipocytes triggered CD36 deacylation and deglycosylation, as well as its dissociation from interacting proteins, prohibitin-1 (PHB) and annexin 2 (ANX2). Our data indicate that lipolysis triggers caveolar endocytosis and translocation of CD36 from the cell membrane to lipid droplets. This study suggests a mechanism for both outside-in and inside-out cellular LCFA transport regulated by CD36 S-acylation and its interactions with PHB and ANX2.
Alexes C. Daquinag, Zhanguo Gao, Cale Fussell, Linnet Immaraj, Renata Pasqualini, Wadih Arap, Askar M. Akimzhanov, Maria Febbraio, Mikhail G. Kolonin
BACKGROUND The aberrant activation of the PI3K/mTOR signaling circuitry is one of the most frequently dysregulated signaling events in head and neck squamous cell carcinoma (HNSCC). Here, we conducted a single-arm, open-label phase IIa clinical trial in individuals with oral premalignant lesions (OPLs) to explore the potential of metformin to target PI3K/mTOR signaling for HNSCC prevention.METHODS Individuals with OPLs, but who were otherwise healthy and without diabetes, underwent pretreatment and posttreatment clinical exam and biopsy. Participants received metformin for 12 weeks (week 1, 500 mg; week 2, 1000 mg; weeks 3–12, 2000 mg daily). Pretreatment and posttreatment biopsies, saliva, and blood were obtained for biomarker analysis, including IHC assessment of mTOR signaling and exome sequencing.RESULTS Twenty-three participants were evaluable for response. The clinical response rate (defined as a ≥50% reduction in lesion size) was 17%. Although lower than the proposed threshold for favorable clinical response, the histological response rate (improvement in histological grade) was 60%, including 17% complete responses and 43% partial responses. Logistic regression analysis revealed that when compared with never smokers, current and former smokers had statistically significantly increased histological responses (P = 0.016). Remarkably, a significant correlation existed between decreased mTOR activity (pS6 IHC staining) in the basal epithelial layers of OPLs and the histological (P = 0.04) and clinical (P = 0.01) responses.CONCLUSION To our knowledge this is the first phase II trial of metformin in individuals with OPLs, providing evidence that metformin administration results in encouraging histological responses and mTOR pathway modulation, thus supporting its further investigation as a chemopreventive agent.TRIAL REGISTRATION NCT02581137FUNDING NIH contract HHSN261201200031I, grants R01DE026644 and R01DE026870
J. Silvio Gutkind, Alfredo A. Molinolo, Xingyu Wu, Zhiyong Wang, Daniela Nachmanson, Olivier Harismendy, Ludmil B. Alexandrov, Beverly R. Wuertz, Frank G. Ondrey, Denise Laronde, Leigha D. Rock, Miriam Rosin, Charles Coffey, Valerie D. Butler, Lisa Bengtson, Chiu-Hsieh Hsu, Julie E. Bauman, Stephen M. Hewitt, Ezra E.W. Cohen, H-H. Sherry Chow, Scott M. Lippman, Eva Szabo
Vascular injury has emerged as a complication contributing to morbidity in coronavirus disease 2019 (COVID-19). The glycosaminoglycan hyaluronan (HA) is a major component of the glycocalyx, a protective layer of glycoconjugates that lines the vascular lumen and regulates key endothelial cell functions. During critical illness, as in the case of sepsis, enzymes degrade the glycocalyx, releasing fragments with pathologic activities into circulation and thereby exacerbating disease. Here, we analyzed levels of circulating glycosaminoglycans in 46 patients with COVID-19 ranging from moderate to severe clinical severity and measured activities of corresponding degradative enzymes. This report provides evidence that the glycocalyx becomes significantly damaged in patients with COVID-19 and corresponds with severity of disease. Circulating HA fragments and hyaluronidase, 2 signatures of glycocalyx injury, strongly associate with sequential organ failure assessment scores and with increased inflammatory cytokine levels in patients with COVID-19. Pulmonary microvascular endothelial cells exposed to COVID-19 milieu show dysregulated HA biosynthesis and degradation, leading to production of pathological HA fragments that are released into circulation. Finally, we show that HA fragments present at high levels in COVID-19 patient plasma can directly induce endothelial barrier dysfunction in a ROCK- and CD44-dependent manner, indicating a role for HA in the vascular pathology of COVID-19.
Kimberly A. Queisser, Rebecca A. Mellema, Elizabeth A. Middleton, Irina Portier, Bhanu Kanth Manne, Frederik Denorme, Ellen J. Beswick, Matthew T. Rondina, Robert A. Campbell, Aaron C. Petrey
Macrophage activation syndrome (MAS) is a life-threatening cytokine storm complicating systemic juvenile idiopathic arthritis (SJIA) driven by IFN-γ. SJIA and MAS are also associated with an unexplained emerging inflammatory lung disease (SJIA-LD), with our recent work supporting pulmonary activation of IFN-γ pathways pathologically linking SJIA-LD and MAS. Our objective was to mechanistically define the potentially novel observation of pulmonary inflammation in the TLR9 mouse model of MAS. In acute MAS, lungs exhibit mild but diffuse CD4-predominant, perivascular interstitial inflammation with elevated IFN-γ, IFN-induced chemokines, and alveolar macrophage (AMϕ) expression of IFN-γ–induced genes. Single-cell RNA sequencing confirmed IFN-driven transcriptional changes across lung cell types with myeloid expansion and detection of MAS-specific macrophage populations. Systemic MAS resolution was associated with increased AMϕ and interstitial lymphocytic infiltration. AMϕ transcriptomic analysis confirmed IFN-γ–induced proinflammatory polarization during acute MAS, which switches toward an antiinflammatory phenotype after systemic MAS resolution. Interestingly, recurrent MAS led to increased alveolar inflammation and lung injury, and it reset AMϕ polarization toward a proinflammatory state. Furthermore, in mice bearing macrophages insensitive to IFN-γ, both systemic features of MAS and pulmonary inflammation were attenuated. These findings demonstrate that experimental MAS induces IFN-γ–driven pulmonary inflammation replicating key features of SJIA-LD and provides a model system for testing potentially novel treatments directed toward SJIA-LD.
Denny K. Gao, Nathan Salomonis, Maggie Henderlight, Christopher Woods, Kairavee Thakkar, Alexei A. Grom, Sherry Thornton, Michael B. Jordan, Kathryn A. Wikenheiser-Brokamp, Grant S. Schulert
Mitochondrial biogenesis and function are controlled by anterograde regulatory pathways involving more than 1000 nuclear-encoded proteins. Transcriptional networks controlling the nuclear-encoded mitochondrial genes remain to be fully elucidated. Here, we show that histone demethylase LSD1 KO from adult mouse liver (LSD1-LKO) reduces the expression of one-third of all nuclear-encoded mitochondrial genes and decreases mitochondrial biogenesis and function. LSD1-modulated histone methylation epigenetically regulates nuclear-encoded mitochondrial genes. Furthermore, LSD1 regulates gene expression and protein methylation of nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), which controls the final step of NAD+ synthesis and limits NAD+ availability in the nucleus. Lsd1 KO reduces NAD+-dependent SIRT1 and SIRT7 deacetylase activity, leading to hyperacetylation and hypofunctioning of GABPβ and PGC-1α, the major transcriptional factor/cofactor for nuclear-encoded mitochondrial genes. Despite the reduced mitochondrial function in the liver, LSD1-LKO mice are protected from diet-induced hepatic steatosis and glucose intolerance, partially due to induction of hepatokine FGF21. Thus, LSD1 orchestrates a core regulatory network involving epigenetic modifications and NAD+ synthesis to control mitochondrial function and hepatokine production.
Yang Cao, Lingyi Tang, Kang Du, Kitt Paraiso, Qiushi Sun, Zhengxia Liu, Xiaolong Ye, Yuan Fang, Fang Yuan, Hank Chen, Yumay Chen, Xiaorong Wang, Clinton Yu, Ira L. Blitz, Ping H. Wang, Lan Huang, Haibo Cheng, Xiang Lu, Ken W.Y. Cho, Marcus Seldin, Zhuyuan Fang, Qin Yang
Aging is associated with chronic oxidative stress and inflammation that affect tissue repair and regeneration capacity. MG53 is a TRIM family protein that facilitates repair of cell membrane injury in a redox-dependent manner. Here, we demonstrate that the expression of MG53 was reduced in failing human hearts and aged mouse hearts, concomitant with elevated NF-κB activation. We evaluated the safety and efficacy of longitudinal, systemic administration of recombinant human MG53 (rhMG53) protein in aged mice. Echocardiography and pressure-volume loop measurements revealed beneficial effects of rhMG53 treatment in improving heart function of aged mice. Biochemical and histological studies demonstrated that the cardioprotective effects of rhMG53 are linked to suppression of NF-κB–mediated inflammation, reducing apoptotic cell death and oxidative stress in the aged heart. Repetitive administration of rhMG53 in aged mice did not have adverse effects on major vital organ functions. These findings support the therapeutic value of rhMG53 in treating age-related decline in cardiac function.
Xiaoliang Wang, Xiuchun Li, Hannah Ong, Tao Tan, Ki Ho Park, Zehua Bian, Xunchang Zou, Erin Haggard, Paul M. Janssen, Robert E. Merritt, Timothy M. Pawlik, Bryan A. Whitson, Nahush A. Mokadam, Lei Cao, Hua Zhu, Chuanxi Cai, Jianjie Ma
Lack of sustained response to therapeutic agents in patients with KRAS-mutant lung cancer poses a major challenge and arises partly due to intratumor heterogeneity that defines phenotypically distinct tumor subpopulations. To attain better therapeutic outcomes, it is important to understand the differential therapeutic sensitivities of tumor cell subsets. Epithelial-mesenchymal transition is a biological phenomenon that can alter the state of cells along a phenotypic spectrum and cause transcriptional rewiring to produce distinct tumor cell subpopulations. We utilized functional shRNA screens, in in vitro and in vivo models, to identify and validate an increased dependence of mesenchymal tumor cells on cyclin-dependent kinase 4 (CDK4) for survival, as well as a mechanism of resistance to MEK inhibitors. High zinc finger E-box binding homeobox 1 levels in mesenchymal tumor cells repressed p21, leading to perturbed CDK4 pathway activity. Increased dependence on CDK4 rendered mesenchymal cancer cells particularly vulnerable to selective CDK4 inhibitors. Coadministration of CDK4 and MEK inhibitors in heterogeneous tumors effectively targeted different tumor subpopulations, subverting the resistance to either single-agent treatment.
Aparna Padhye, Jessica M. Konen, B. Leticia Rodriguez, Jared J. Fradette, Joshua K. Ochieng, Lixia Diao, Jing Wang, Wei Lu, Luisa S. Solis, Harsh Batra, Maria G. Raso, Michael D. Peoples, Rosalba Minelli, Alessandro Carugo, Christopher A. Bristow, Don L. Gibbons
Immunotherapies are needed in the clinic that effectively suppress β cell autoimmunity and reestablish long-term self-tolerance in type 1 diabetes. We previously demonstrated that nondepleting anti-CD4 (αCD4) and αCD8α antibodies establish rapid and indefinite remission in recent-onset diabetic NOD mice. Diabetes reversal by coreceptor therapy (CoRT) is induced by suppression of pathogenic effector T cells (Teffs) and the selective egress of T cells from the pancreatic lymph nodes and islets that remain free of infiltration in the long term. Here, we defined CoRT-induced events regulating early Teff function and pancreatic residency, and long-term tolerance. TCR-driven gene expression controlling autoreactive Teff expansion and proinflammatory activity was suppressed by CoRT, and islet T cell egress was dependent on sphingosine-1 phosphate. In both murine and human T cells, CoRT upregulated the Foxo1 transcriptional axis, which in turn was required for suppression and efficient pancreatic egress of Teffs. Interestingly, long-term tolerance induced in late-preclinical NOD mice was marked by reseeding of the pancreas by a reduced CD8+ Teff pool exhibiting an exhausted phenotype. Notably, PD-1 blockade, which rescues exhausted Teffs, resulted in diabetes onset in protected animals. These findings demonstrate that CoRT has distinct intrinsic effects on Teffs that impact events early in induction and later in maintenance of self-tolerance.
Matthew Clark, Charles J. Kroger, Qi Ke, Rui Zhang, Karen Statum, J. Justin Milner, Aaron Martin, Bo Wang, Roland Tisch
Neutrophil-mediated activation and injury of the endothelium play roles in the pathogenesis of diverse disease states ranging from autoimmunity to cancer to COVID-19. Neutralization of cationic proteins (such as neutrophil extracellular trap–derived [NET-derived] histones) with polyanionic compounds has been suggested as a potential strategy for protecting the endothelium from such insults. Here, we report that the US Food and Drug Administration–approved polyanionic agent defibrotide (a pleiotropic mixture of oligonucleotides) directly engages histones and thereby blocks their pathological effects on endothelium. In vitro, defibrotide counteracted endothelial cell activation and pyroptosis-mediated cell death, whether triggered by purified NETs or recombinant histone H4. In vivo, defibrotide stabilized the endothelium and protected against histone-accelerated inferior vena cava thrombosis in mice. Mechanistically, defibrotide demonstrated direct and tight binding to histone H4 as detected by both electrophoretic mobility shift assay and surface plasmon resonance. Taken together, these data provide insights into the potential role of polyanionic compounds in protecting the endothelium from thromboinflammation with potential implications for myriad NET- and histone-accelerated disease states.
Hui Shi, Alex A. Gandhi, Stephanie A. Smith, Qiuyu Wang, Diane Chiang, Srilakshmi Yalavarthi, Ramadan A. Ali, Chao Liu, Gautam Sule, Pei-Suen Tsou, Yu Zuo, Yogendra Kanthi, Evan A. Farkash, Jiandie D. Lin, James H. Morrissey, Jason S. Knight
Glioblastoma (GBM) is characterized by an aberrant yet druggable epigenetic landscape. One major family of epigenetic regulators, the histone deacetylases (HDACs), are considered promising therapeutic targets for GBM due to their repressive influences on transcription. Although HDACs share redundant functions and common substrates, the unique isoform-specific roles of different HDACs in GBM remain unclear. In neural stem cells, HDAC2 is the indispensable deacetylase to ensure normal brain development and survival in the absence of HDAC1. Surprisingly, we find that HDAC1 is the essential class I deacetylase in glioma stem cells, and its loss is not compensated for by HDAC2. Using cell-based and biochemical assays, transcriptomic analyses, and patient-derived xenograft models, we find that knockdown of HDAC1 alone has profound effects on the glioma stem cell phenotype in a p53-dependent manner. We demonstrate marked suppression in tumor growth upon targeting of HDAC1 and identify compensatory pathways that provide insights into combination therapies for GBM. Our study highlights the importance of HDAC1 in GBM and the need to develop isoform-specific drugs.
Costanza Lo Cascio, James B. McNamara, Ernesto L. Melendez, Erika M. Lewis, Matthew E. Dufault, Nader Sanai, Christopher L. Plaisier, Shwetal Mehta
Lipin 1 regulates cellular lipid homeostasis through roles in glycerolipid synthesis (through phosphatidic acid phosphatase activity) and transcriptional coactivation. Lipin 1–deficient individuals exhibit episodic disease symptoms that are triggered by metabolic stress, such as stress caused by prolonged fasting. We sought to identify critical lipin 1 activities during fasting. We determined that lipin 1 deficiency induces widespread alternative mRNA splicing in liver during fasting, much of which is normalized by refeeding. The role of lipin 1 in mRNA splicing was largely independent of its enzymatic function. We identified interactions between lipin 1 and spliceosome proteins, as well as a requirement for lipin 1 to maintain homeostatic levels of spliceosome small nuclear RNAs and specific RNA splicing factors. In fasted Lpin1–/– liver, we identified a correspondence between alternative splicing of phospholipid biosynthetic enzymes and dysregulated phospholipid levels; splicing patterns and phospholipid levels were partly normalized by feeding. Thus, lipin 1 influences hepatic lipid metabolism through mRNA splicing, as well as through enzymatic and transcriptional activities, and fasting exacerbates the deleterious effects of lipin 1 deficiency on metabolic homeostasis.
Huan Wang, Tracey W. Chan, Ajay A. Vashisht, Brian G. Drew, Anna C. Calkin, Thurl E. Harris, James A. Wohlschlegel, Xinshu Xiao, Karen Reue
BACKGROUND Targeted arterial infusion of verapamil combined with chemotherapy (TVCC) is an effective clinical interventional therapy for esophageal squamous cell carcinoma (ESCC), but multidrug resistance (MDR) remains the major cause of relapse or poor prognosis, and the underlying molecular mechanisms of MDR, temporal intratumoral heterogeneity, and clonal evolutionary processes of resistance have not been determined.METHODS To elucidate the roles of genetic and epigenetic alterations in the evolution of acquired resistance during therapies, we performed whole-exome sequencing on 16 serial specimens from 7 patients with ESCC at every cycle of therapeutic intervention from 3 groups, complete response, partial response, and progressive disease, and we performed whole-genome bisulfite sequencing for 3 of these 7 patients, 1 patient from each group.RESULTS Patients with progressive disease exhibited a substantially higher genomic and epigenomic temporal heterogeneity. Subclonal expansions driven by the beneficial new mutations were observed during combined therapies, which explained the emergence of MDR. Notably, SLC7A8 was identified as a potentially novel MDR gene, and functional assays demonstrated that mutant SLC7A8 promoted the resistance phenotypes of ESCC cell lines. Promoter methylation dynamics during treatments revealed 8 drug resistance protein-coding genes characterized by hypomethylation in promoter regions. Intriguingly, promoter hypomethylation of SLC8A3 and mutant SLC7A8 were enriched in an identical pathway, protein digestion and absorption, indicating a potentially novel MDR mechanism during treatments.CONCLUSION Our integrated multiomics investigations revealed the dynamics of temporal genetic and epigenetic inter- and intratumoral heterogeneity, clonal evolutionary processes, and epigenomic changes, providing potential MDR therapeutic targets in treatment-resistant patients with ESCC during combined therapies.FUNDING National Natural Science Foundation of China, Science Foundation of Peking University Cancer Hospital, CAMS Innovation Fund for Medical Sciences, Major Program of Shenzhen Bay Laboratory, Guangdong Basic and Applied Basic Research Foundation, and the third round of public welfare development and reform pilot projects of Beijing Municipal Medical Research Institutes.
Qingjie Min, Yan Wang, Qingnan Wu, Xianfeng Li, Huajing Teng, Jiawen Fan, Yiren Cao, Pingsheng Fan, Qimin Zhan
A dynamically regulated microenvironment, which is mediated by crosstalk between adipocytes and neighboring cells, is critical for adipose tissue homeostasis and function. However, information on key molecules and/or signaling pathways regulating the crosstalk remains limited. In this study, we identify adipocyte miRNA-182-5p (miR-182-5p) as a crucial antiobesity molecule that stimulated beige fat thermogenesis by promoting the crosstalk between adipocytes and macrophages. miR-182-5p was highly enriched in thermogenic adipocytes, and its expression was markedly stimulated by cold exposure in mice. In contrast, miR-182-5p expression was significantly reduced in adipose tissues of obese humans and mice. Knockout of miR-185-5p decreased cold-induced beige fat thermogenesis whereas overexpression of miR-185-5p increased beiging and thermogenesis in mice. Mechanistically, miR-182-5p promoted FGF21 expression and secretion in adipocytes by suppressing nuclear receptor subfamily 1 group D member 1 (Nr1d1) at 5′-UTR, which in turn stimulates acetylcholine synthesis and release in macrophages. Increased acetylcholine expression activated the nicotine acetylcholine receptor in adipocytes, which stimulated PKA signaling and consequent thermogenic gene expression. Our study reveals a key role of the miR-182-5p/FGF21/acetylcholine/acetylcholine receptor axis that mediates the crosstalk between adipocytes and macrophages to promote beige fat thermogenesis. Activation of the miR-182-5p–induced signaling pathway in adipose tissue may be an effective approach to ameliorate obesity and associated metabolic diseases.
Wen Meng, Ting Xiao, Xiuci Liang, Jie Wen, Xinyi Peng, Jing Wang, Yi Zou, Jiahao Liu, Christie Bialowas, Hairong Luo, Yacheng Zhang, Bilian Liu, Jingjing Zhang, Fang Hu, Meilian Liu, Lily Q. Dong, Zhiguang Zhou, Feng Liu, Juli Bai
Retinoic acid (RA) signaling has long been speculated to regulate embryo implantation, because many enzymes and proteins responsible for maintaining RA homeostasis and transducing RA signals are tightly regulated in the endometrium during this critical period. However, due to a lack of genetic data, it was unclear whether RA signaling is truly required for implantation and which specific RA signaling cascades are at play. Herein we utilize a genetic murine model that expresses a dominant-negative form of RA receptor (RAR) specifically in female reproductive organs to show that functional RA signaling is fundamental to female fertility, particularly implantation and decidualization. Reduction in RA signaling activity severely affects the ability of the uterus to achieve receptive status and decidualize, partially through dampening follistatin expression and downstream activin B/bone morphogenetic protein 2 signaling. To confirm translational relevance of these findings to humans, human endometrial stromal cells (hESCs) were treated with a pan-RAR antagonist to show that in vitro decidualization is impaired. RNA interference perturbation of individual RAR transcripts in hESCs revealed that RARα in particular was essential for proper decidualization. These data provide direct functional evidence that uterine RAR-mediated RA signaling was crucial for mammalian embryo implantation, and its disruption led to failure of uterine receptivity and decidualization, resulting in severely compromised fertility.
Yan Yin, Meade E. Haller, Sangappa B. Chadchan, Ramakrishna Kommagani, Liang Ma
Host genes define the severity of inflammation and immunity but specific loci doing so are unknown. Here we show that TNF receptor superfamily member 13B (TNFRSF13B) variants, which enhance defense against certain pathogens, also control immune-mediated injury of transplants, by regulating innate B cells’ functions. Analysis of TNFRSF13B in human kidney transplant recipients revealed that 33% of those with antibody-mediated rejection (AMR) but fewer than 6% of those with stable graft function had TNFRSF13B missense mutations. To explore mechanisms underlying aggressive immune responses, we investigated alloimmunity and rejection in mice. Cardiac allografts in Tnfrsf13b-mutant mice underwent early and severe AMR. The dominance and precocity of AMR in Tnfrsf13b-deficient mice were not caused by increased alloantibodies. Rather, Tnfrsf13b mutations decreased “natural” IgM and compromised complement regulation, leading to complement deposition in allografted hearts and autogenous kidneys. Thus, WT TNFRSF13B and Tnfrsf13b support innate B cell functions that limit complement-associated inflammation; in contrast, common variants of these genes intensify inflammatory responses that help clear microbial infections but allow inadvertent tissue injury to ensue. The wide variation in inflammatory reactions associated with TNFRSF13B diversity suggests polymorphisms could underlie variation in host defense and explosive inflammatory responses that sometimes enhance morbidity associated with immune responses.
Mayara Garcia de Mattos Barbosa, Adam R. Lefferts, Daniel Huynh, Hui Liu, Yu Zhang, Beverly Fu, Jenna Barnes, Milagros Samaniego, Richard J. Bram, Raif S. Geha, Ariella Shikanov, Eline T. Luning Prak, Evan A. Farkash, Jeffrey L. Platt, Marilia Cascalho
The metabolic environment is important for neuronal cells, such as photoreceptors. When photoreceptors undergo degeneration, as occurs during retinitis pigmentosa (RP), patients have progressive loss of vision that proceeds to full blindness. Currently, there are no available treatments for the majority of RP diseases. We performed metabolic profiling of the neural retina in a preclinical model of RP and found that TCA cycle intermediates were reduced during disease. We then determined that (a) promoting citrate production within the TCA cycle in retinal neurons during disease progression protected the photoreceptors from cell death and prolonged visual function, (b) supplementation with single metabolites within the TCA cycle provided this therapeutic effect in vivo over time, and (c) this therapeutic effect was not specific to a particular genetic mutation but had broad applicability for patients with RP and other retinal degenerative diseases. Overall, targeting TCA cycle activity in the neural retina promoted photoreceptor survival and visual function during neurodegenerative disease.
Ashley A. Rowe, Pinkal D. Patel, Ruth Gordillo, Katherine J. Wert
As SARS-CoV-2 continues to spread globally, questions have emerged regarding the strength and durability of immune responses in specific populations. In this study, we evaluated humoral immune responses in 69 children and adolescents with asymptomatic or mild symptomatic SARS-CoV-2 infection. We detected robust IgM, IgG, and IgA antibody responses to a broad array of SARS-CoV-2 antigens at the time of acute infection and 2 and 4 months after acute infection in all participants. Notably, these antibody responses were associated with virus-neutralizing activity that was still detectable 4 months after acute infection in 94% of children. Moreover, antibody responses and neutralizing activity in sera from children and adolescents were comparable or superior to those observed in sera from 24 adults with mild symptomatic infection. Taken together, these findings indicate that children and adolescents with mild or asymptomatic SARS-CoV-2 infection generate robust and durable humoral immune responses that can likely contribute to protection from reinfection.
Carolina Garrido, Jillian H. Hurst, Cynthia G. Lorang, Jhoanna N. Aquino, Javier Rodriguez, Trevor S. Pfeiffer, Tulika Singh, Eleanor C. Semmes, Debra J. Lugo, Alexandre T. Rotta, Nicholas A. Turner, Thomas W. Burke, Micah T. McClain, Elizabeth A. Petzold, Sallie R. Permar, M. Anthony Moody, Christopher W. Woods, Matthew S. Kelly, Genevieve G. Fouda
The NR4A family of orphan nuclear receptors (Nr4a1–3) plays redundant roles to establish and maintain Treg identity; deletion of multiple family members in the thymus results in Treg deficiency and a severe inflammatory disease. Consequently, it has been challenging to unmask redundant functions of the NR4A family in other immune cells. Here we use a competitive bone marrow chimera strategy, coupled with conditional genetic tools, to rescue Treg homeostasis and unmask such functions. Unexpectedly, chimeras harboring Nr4a1–/– Nr4a3–/– (double-knockout, DKO) bone marrow developed autoantibodies and a systemic inflammatory disease despite a replete Treg compartment of largely WT origin. This disease differs qualitatively from that seen with Treg deficiency and is B cell extrinsic. Negative selection of DKO thymocytes is profoundly impaired in a cell-intrinsic manner. Consistent with escape of self-reactive T cells into the periphery, DKO T cells with functional, phenotypic, and transcriptional features of anergy accumulated in chimeric mice. Nevertheless, we observed upregulation of genes encoding inflammatory mediators in anergic DKO T cells, and DKO T cells exhibited enhanced capacity for IL-2 production. These studies reveal cell-intrinsic roles for the NR4A family in both central and peripheral T cell tolerance and demonstrate that each is essential to preserve immune homeostasis.
Ryosuke Hiwa, Hailyn V. Nielsen, James L. Mueller, Ravi Mandla, Julie Zikherman
Status epilepticus (SE) is a neurological emergency usually accompanied by acute cerebral edema and long-term cognitive impairment, and is characterized by neurodegeneration and aberrant hyperphosphorylated tau protein (p-tau) aggregation. The glia-lymphatic (glymphatic) system plays a central role in facilitating the clearance of metabolic waste from the brain, but its relationship with cerebral edema and cognitive dysfunction after SE is unclear. We hypothesized that cerebral edema after SE might impair glymphatic system function through compression, thus leading to impaired removal of metabolic waste, and ultimately affecting long-term cognitive function. Our results showed that glymphatic system function was temporarily impaired, as evidenced by 2-photon imaging, MRI enhancement, imaging of brain sections, and astrocytic water channel aquaporin 4 (AQP4) protein polarization. The severity of cerebral edema on MRI correlated well with glymphatic system dysfunction within 8 days following SE. Moreover, when cerebral edema was alleviated by glibenclamide treatment or genetic deletion of Trpm4, post-SE glymphatic system function recovered earlier, along with fewer p-tau–deposited neurons and neuronal degeneration and better cognitive function. These findings suggest that SE-induced cerebral edema may cause glymphatic system dysfunction and render the post-SE brain vulnerable to p-tau aggregation and neurocognitive impairment.
Kewei Liu, Juan Zhu, Yuan Chang, Zhenzhou Lin, Zhu Shi, Xing Li, Xing Chen, Chuman Lin, Suyue Pan, Kaibin Huang