Mitochondrial quality control (MQC) is crucial for regulating CNS homeostasis, and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human postmortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide therapeutic options for the management of DR-associated complications.
Jose R. Hombrebueno, Lauren Cairns, Louise R. Dutton, Timothy J. Lyons, Derek P. Brazil, Paul Moynagh, Tim M. Curtis, Heping Xu
At diagnosis, most people with type 1 diabetes (T1D) produce measurable levels of endogenous insulin, but the rate at which insulin secretion declines is heterogeneous. To explain this heterogeneity, we sought to identify a composite signature predictive of insulin secretion, using a collaborative assay evaluation and analysis pipeline that incorporated multiple cellular and serum measures reflecting β cell health and immune system activity. The ability to predict decline in insulin secretion would be useful for patient stratification for clinical trial enrollment or therapeutic selection. Analytes from 12 qualified assays were measured in shared samples from subjects newly diagnosed with T1D. We developed a computational tool (DIFAcTO, Data Integration Flexible to Account for different Types of data and Outcomes) to identify a composite panel associated with decline in insulin secretion over 2 years following diagnosis. DIFAcTO uses multiple filtering steps to reduce data dimensionality, incorporates error estimation techniques including cross-validation and sensitivity analysis, and is flexible to assay type, clinical outcome, and disease setting. Using this novel analytical tool, we identified a panel of immune markers that, in combination, are highly associated with loss of insulin secretion. The methods used here represent a potentially novel process for identifying combined immune signatures that predict outcomes relevant for complex and heterogeneous diseases like T1D.
Cate Speake, Samuel O. Skinner, Dror Berel, Elizabeth Whalen, Matthew J. Dufort, William Chad Young, Jared M. Odegard, Anne M. Pesenacker, Frans K. Gorus, Eddie A. James, Megan K. Levings, Peter S. Linsley, Eitan M. Akirav, Alberto Pugliese, Martin J. Hessner, Gerald T. Nepom, Raphael Gottardo, S. Alice Long
Muscle contractures are a prominent and disabling feature of many neuromuscular disorders, including the 2 most common forms of childhood neurologic dysfunction: neonatal brachial plexus injury (NBPI) and cerebral palsy. There are currently no treatment strategies to directly alter the contracture pathology, as the pathogenesis of these contractures is unknown. We previously showed in a mouse model of NBPI that contractures result from impaired longitudinal muscle growth. Current presumed explanations for growth impairment in contractures focus on the dysregulation of muscle stem cells, which differentiate and fuse to existing myofibers during growth, as this process has classically been thought to control muscle growth during the neonatal period. Here, we demonstrate in a mouse model of NBPI that denervation does not prevent myonuclear accretion and that reduction in myonuclear number has no effect on functional muscle length or contracture development, providing definitive evidence that altered myonuclear accretion is not a driver of neuromuscular contractures. In contrast, we observed elevated levels of protein degradation in NBPI muscle, and we demonstrate that contractures can be pharmacologically prevented with the proteasome inhibitor bortezomib. These studies provide what we believe is the first strategy to prevent neuromuscular contractures by correcting the underlying deficit in longitudinal muscle growth.
Sia Nikolaou, Alyssa A.W. Cramer, Liangjun Hu, Qingnian Goh, Douglas P. Millay, Roger Cornwall
The choroid plexus (ChP) is a highly vascularized tissue found in the brain ventricles, with an apical epithelial cell layer surrounding fenestrated capillaries. It is responsible for the production of most of the cerebrospinal fluid (CSF) in the ventricular system, subarachnoid space, and central canal of the spinal cord, while also constituting the blood-CSF barrier (BCSFB). In addition, epithelial cells of the ChP (EChP) synthesize neurotrophic factors and other signaling molecules that are released into the CSF. Here, we show that insulin is produced in EChP of mice and humans, and its expression and release are regulated by serotonin. Insulin mRNA and immune-reactive protein, including C-peptide, are present in EChP, as detected by several experimental approaches, and appear in much higher levels than any other brain region. Moreover, insulin is produced in primary cultured mouse EChP, and its release, albeit Ca2+ sensitive, is not regulated by glucose. Instead, activation of the 5HT2C receptor by serotonin treatment led to activation of IP3-sensitive channels and Ca2+ mobilization from intracellular storage, leading to insulin secretion. In vivo depletion of brain serotonin in the dorsal raphe nucleus negatively affected insulin expression in the ChP, suggesting an endogenous modulation of ChP insulin by serotonin. Here, we show for the first time to our knowledge that insulin is produced by EChP in the brain, and its release is modulated at least by serotonin but not glucose.
Caio Henrique Mazucanti, Qing-Rong Liu, Doyle Lang, Nicholas Huang, Jennifer F. O’Connell, Simonetta Camandola, Josephine M. Egan
Mice homozygous for a hypomorphic allele of DNA replication factor minichromosome maintenance protein 2 (designated Mcm2cre/cre) develop precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL) with 4–32 small interstitial deletions per tumor. Mice that express a NUP98-HOXD13 (NHD13) transgene develop multiple types of leukemia, including myeloid and T and B lymphocyte. All Mcm2cre/cre NHD13+ mice develop pre-T LBL, and 26% develop an unrelated, concurrent B cell precursor acute lymphoblastic leukemia (BCP-ALL). Copy number alteration (CNA) analysis demonstrated that pre-T LBLs were characterized by homozygous deletions of Pten and Tcf3 and partial deletions of Notch1 leading to Notch1 activation. In contrast, BCP-ALLs were characterized by recurrent deletions involving Pax5 and Ptpn1 and copy number gain of Abl1 and Nup214 resulting in a Nup214-Abl1 fusion. We present a model in which Mcm2 deficiency leads to replicative stress, DNA double strand breaks (DSBs), and resultant CNAs due to errors in DNA DSB repair. CNAs that involve critical oncogenic pathways are then selected in vivo as malignant lymphoblasts because of a fitness advantage. Some CNAs, such as those involving Abl1 and Notch1, represent attractive targets for therapy.
Mianmian Yin, Timour Baslan, Robert L. Walker, Yuelin J. Zhu, Amy Freeland, Toshihiro Matsukawa, Sriram Sridharan, André Nussenzweig, Steven C. Pruitt, Scott W. Lowe, Paul S. Meltzer, Peter D. Aplan
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on endoplasmic reticulum (ER) stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction, and hypertrophic remodeling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production, and cytoskeleton-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signaling in CADASIL were ameliorated by inhibitors of Notch3 (γ-secretase inhibitor), Nox5 (mellitin), ER stress (4-phenylbutyric acid), and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation–associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ER stress/ROCK signaling as a putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.
Karla B. Neves, Adam P. Harvey, Fiona Moreton, Augusto C. Montezano, Francisco J. Rios, Rhéure Alves-Lopes, Aurelie Nguyen Dinh Cat, Paul Rocchicciolli, Christian Delles, Anne Joutel, Keith Muir, Rhian M. Touyz
Gonorrhea is a sexually transmitted infection with 87 million new cases per year globally. Increasing antibiotic resistance has severely limited treatment options. A mechanism that Neisseria gonorrhoeae uses to evade complement attack is binding of the complement inhibitor C4b-binding protein (C4BP). We screened 107 porin B1a (PorB1a) and 83 PorB1b clinical isolates randomly selected from a Swedish strain collection over the last 10 years and noted that 96/107 (89.7%) PorB1a and 16/83 (19.3%) PorB1b bound C4BP; C4BP binding substantially correlated with the ability to evade complement-dependent killing (r = 0.78). We designed 2 chimeric proteins that fused C4BP domains to the backbone of IgG or IgM (C4BP-IgG; C4BP-IgM) with the aim of enhancing complement activation and killing of gonococci. Both proteins bound gonococci (KD C4BP-IgM = 2.4 nM; KD C4BP-IgG 980.7 nM), but only hexameric C4BP-IgM efficiently outcompeted heptameric C4BP from the bacterial surface, resulting in enhanced complement deposition and bacterial killing. Furthermore, C4BP-IgM substantially attenuated the duration and burden of colonization of 2 C4BP-binding gonococcal isolates but not a non–C4BP-binding strain in a mouse vaginal colonization model using human factor H/C4BP–transgenic mice. Our preclinical data present C4BP-IgM as an adjunct to conventional antimicrobials for the treatment of gonorrhea.
Serena Bettoni, Jutamas Shaughnessy, Karolina Maziarz, David Ermert, Sunita Gulati, Bo Zheng, Matthias Mörgelin, Susanne Jacobsson, Kristian Riesbeck, Magnus Unemo, Sanjay Ram, Anna M. Blom
Pulmonary drug delivery presents a unique opportunity to target lower airway inflammation, which is often characterized by the massive recruitment of neutrophils from blood. However, specific therapies are lacking modulation of airway neutrophil function, and difficult challenges must be overcome to achieve therapeutic efficacy against pulmonary inflammation, notably drug hydrophobicity, mucociliary and macrophage-dependent clearance, and high extracellular protease burden. Here, we present a multistage, aerodynamically favorable delivery platform that uses extracellular proteolysis to its advantage to deliver nanoparticle-embedded hydrophobic drugs to neutrophils within the lower airways. Our design consists of a self-regulated nanoparticle-in-microgel system, in which microgel activation is triggered by extracellular elastase (degranulated by inflammatory neutrophils), and nanoparticles are loaded with Nexinhib20, a potent neutrophil degranulation inhibitor. Successful in vivo delivery of Nexinhib20 to the airways and into neutrophils promoted resolution of the inflammatory response by dampening neutrophil recruitment and degranulation, proinflammatory cytokine production in both airway and systemic compartments, as well as the presence of neutrophil-derived pathological extracellular vesicles in the lung fluid. Our findings showcase a new platform that overcomes challenges in pulmonary drug delivery and allows customization to match the proteolytic footprint of given diseases.
Joscelyn C. Mejías, Osric A. Forrest, Camilla Margaroli, David A. Frey Rubio, Liliana Viera, Jindong Li, Xin Xu, Amit Gaggar, Rabindra Tirouvanziam, Krishnendu Roy
Mitophagy, by maintaining mitochondrial quality control, plays a key role in maintaining kidney function and is impaired in pathologic states. Macrophages are well known for their pathogenic role in kidney fibrosis. Here, we report that PINK1/Parkin-mediated mitophagy in macrophages is compromised in experimental and human kidney fibrosis. We demonstrate downregulation of mitophagy regulators mitofusin-2 (MFN2) and Parkin downstream of PINK1 in kidney fibrosis. Loss of either Pink1 or Prkn promoted renal extracellular matrix accumulation and frequency of profibrotic/M2 macrophages. Pink1–/– or Prkn–/– BM-derived macrophages (BMDMs) showed enhanced expression of rictor. Mitochondria from TGF-β1–treated Pink1–/– BMDMs exhibited increased superoxide levels, along with reduced respiration and ATP production. In addition, mitophagy in macrophages involves PINK1-mediated phosphorylation of downstream MFN2, MFN2-facilitated recruitment of Parkin to damaged mitochondria, and macrophage-specific deletion of Mfn2 aggravates kidney fibrosis. Moreover, mitophagy regulators were downregulated in human CKD kidney and TGF-β1–treated human renal macrophages, whereas Mdivi1 treatment suppressed mitophagy mediators and promoted fibrotic response. Taken together, our study is the first to our knowledge to demonstrate that macrophage mitophagy plays a protective role against kidney fibrosis via regulating the PINK1/MFN2/Parkin-mediated pathway.
Divya Bhatia, Kuei-Pin Chung, Kiichi Nakahira, Edwin Patino, Michelle C. Rice, Lisa K. Torres, Thangamani Muthukumar, Augustine M.K. Choi, Oleh M. Akchurin, Mary E. Choi
BACKGROUND The presence of an early repolarization pattern (ERP) on the surface ECG is associated with risk of ventricular fibrillation and sudden cardiac death. Family studies have shown that ERP is a highly heritable trait, but molecular genetic determinants are unknown.METHODS To identify genetic susceptibility loci for ERP, we performed a GWAS and meta-analysis in 2,181 cases and 23,641 controls of European ancestry.RESULTS We identified a genome-wide significant (P < 5 × 10–8) locus in the potassium voltage-gated channel subfamily D member 3 (KCND3) gene that was successfully replicated in additional 1,124 cases and 12,510 controls. A subsequent joint meta-analysis of the discovery and replication cohorts identified rs1545300 as the lead SNP at the KCND3 locus (OR 0.82 per minor T allele, P = 7.7 × 10–12) but did not reveal additional loci. Colocalization analyses indicate causal effects of KCND3 gene expression levels on ERP in both cardiac left ventricle and tibial artery.CONCLUSIONS In this study, we identified for the first time to our knowledge a genome-wide significant association of a genetic variant with ERP. Our findings of a locus in the KCND3 gene provide insights not only into the genetic determinants but also into the pathophysiological mechanism of ERP, discovering a promising candidate for functional studies.FUNDING This project was funded by the German Center for Cardiovascular Research (DZHK Shared Expertise SE081 – STATS). For detailed funding information per study, see the Supplemental Acknowledgments.
Alexander Teumer, Teresa Trenkwalder, Thorsten Kessler, Yalda Jamshidi, Marten E. van den Berg, Bernhard Kaess, Christopher P. Nelson, Rachel Bastiaenen, Marzia De Bortoli, Alessandra Rossini, Isabel Deisenhofer, Klaus Stark, Solmaz Assa, Peter S. Braund, Claudia Cabrera, Anna F. Dominiczak, Martin Gögele, Leanne M. Hall, M. Arfan Ikram, Maryam Kavousi, Karl J. Lackner, Lifelines Cohort Study, Christian Müller, Thomas Münzel, Matthias Nauck, Sandosh Padmanabhan, Norbert Pfeiffer, Tim D. Spector, Andre G. Uitterlinden, Niek Verweij, Uwe Völker, Helen R. Warren, Mobeen Zafar, Stephan B. Felix, Jan A. Kors, Harold Snieder, Patricia B. Munroe, Cristian Pattaro, Christian Fuchsberger, Georg Schmidt, Ilja M. Nolte, Heribert Schunkert, Peter P. Pramstaller, Philipp S. Wild, Pim van der Harst, Bruno H. Stricker, Renate B. Schnabel, Nilesh J. Samani, Christian Hengstenberg, Marcus Dörr, Elijah R. Behr, Wibke Reinhard
The purpose of this study was to define the mechanism by which cardiac neuraxial decentralization or spinal cord stimulation (SCS) reduces ischemia-induced ventricular fibrillation (VF). Direct measurements of norepinephrine (NE) levels in the left ventricular interstitial fluid (ISF) by microdialysis, in response to transient (15-minute) coronary artery occlusion (CAO), were performed in anesthetized canines. Responses were studied in animals with intact neuraxes and were compared with those in which the intrathoracic component of the cardiac neuraxes (stellate ganglia) or the intrinsic cardiac neuronal (ICN) system was surgically delinked from the central nervous system and those with intact neuraxes with preemptive SCS (T1–T3). With intact neuraxes, animals with exaggerated NE release due to CAO were at increased risk for VF. During CAO, there was a 152% increase in NE when the neuraxes were intact compared with 114% following stellate decentralization and 16% following ICN decentralization. During SCS, CAO NE levels increased by 59%. Risk for CAO-induced VF was 38% in controls, 8% following decentralization, and 11% following SCS. These data indicate that ischemia-related afferent neuronal transmission differentially engages central and intrathoracic sympathetic reflexes and amplifies sympathoexcitation. Differences in regional ventricular NE release are associated with increased risk for VF. Surgical decentralization or SCS reduced NE release and VF.
Jeffrey L. Ardell, Robert D. Foreman, J. Andrew Armour, Kalyanam Shivkumar
Diabetic foot ulcers (DFUs) are a life-threatening disease that often results in lower limb amputations and a shortened life span. Current treatment options are limited and often not efficacious, raising the need for new therapies. To investigate the therapeutic potential of topical statins to restore healing in patients with DFUs, we performed next-generation sequencing on mevastatin-treated primary human keratinocytes. We found that mevastatin activated and modulated the EGF signaling to trigger an antiproliferative and promigratory phenotype, suggesting that statins may shift DFUs from a hyperproliferative phenotype to a promigratory phenotype in order to stimulate healing. Furthermore, mevastatin induced a migratory phenotype in primary human keratinocytes through EGF-mediated activation of Rac1, resulting in actin cytoskeletal reorganization and lamellipodia formation. Interestingly, the EGF receptor is downregulated in tissue biopsies from patients with DFUs. Mevastatin restored EGF signaling in DFUs through disruption of caveolae to promote keratinocyte migration, which was confirmed by caveolin-1 (Cav1) overexpression studies. We conclude that topical statins may have considerable therapeutic potential as a treatment option for patients with DFUs and offer an effective treatment for chronic wounds that can be rapidly translated to clinical use.
Andrew P. Sawaya, Ivan Jozic, Rivka C. Stone, Irena Pastar, Andjela N. Egger, Olivera Stojadinovic, George D. Glinos, Robert S. Kirsner, Marjana Tomic-Canic
Angiogenesis is a key process that allows nutrient uptake and cellular trafficking and is coopted in cancer to enable tumor growth and metastasis. Recently, extracellular vesicles (EVs) have been shown to promote angiogenesis; however, it is unclear what unique features EVs contribute to the process. Here, we studied the role of EVs derived from head and neck squamous cell carcinoma (HNSCC) in driving tumor angiogenesis. Small EVs (SEVs), in the size range of exosomes (50–150 nm), induced angiogenesis both in vitro and in vivo. Proteomic analysis of HNSCC SEVs revealed the cell-to-cell signaling receptor ephrin type B receptor 2 (EPHB2) as a promising candidate cargo to promote angiogenesis. Analysis of patient data further identified EPHB2 overexpression in HNSCC tumors to be associated with poor patient prognosis and tumor angiogenesis, especially in the context of overexpression of the exosome secretion regulator cortactin. Functional experiments revealed that EPHB2 expression in SEVs regulated angiogenesis both in vitro and in vivo and that EPHB2 carried by SEVs stimulates ephrin-B reverse signaling, inducing STAT3 phosphorylation. A STAT3 inhibitor greatly reduced SEV-induced angiogenesis. These data suggest a model in which EVs uniquely promote angiogenesis by transporting Eph transmembrane receptors to nonadjacent endothelial cells to induce ephrin reverse signaling.
Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver
Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case-control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman’s rho, P = 6 × 10–8). A CMV-specific IFN-γ response was also associated with increased risk of developing TB disease (conditional logistic regression; P = 0.043; OR, 2.2; 95% CI, 1.02–4.83) and shorter time to TB diagnosis (Log Rank Mantel-Cox, P = 0.037). CMV+ infants who developed TB disease had lower expression of NK cell–associated gene signatures and a lower frequency of CD3–CD4–CD8– lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot–positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and –negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV– signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.
Julius Müller, Rachel Tanner, Magali Matsumiya, Margaret A. Snowden, Bernard Landry, Iman Satti, Stephanie A. Harris, Matthew K. O’Shea, Lisa Stockdale, Leanne Marsay, Agnieszka Chomka, Rachel Harrington-Kandt, Zita-Rose Manjaly Thomas, Vivek Naranbhai, Elena Stylianou, Stanley Kimbung Mbandi, Mark Hatherill, Gregory Hussey, Hassan Mahomed, Michele Tameris, J. Bruce McClain, Thomas G. Evans, Willem A. Hanekom, Thomas J. Scriba, Helen McShane, Helen A. Fletcher
Dietary salt consumption leads to cutaneous Na+ storage and is associated with various disorders, including osteopenia. Here, we explore the impact of Na+ and the osmoprotective transcription factor nuclear factor of activated T cell 5 (NFAT5) on bone density and osteoclastogenesis. Compared with treatment of mice with high-salt diet, low-salt diet (LSD) increased bone density, decreased osteoclast numbers, and elevated Na+ content and Nfat5 levels in the BM. This response to LSD was dependent on NFAT5 expressed in myeloid cells. Simulating in vivo findings, we exposed osteoclast precursors and osteoblasts to elevated Na+ content (high-salt conditions; HS¢), resulting in increased NFAT5 binding to the promotor region of RANKL decoy receptor osteoprotegerin (OPG). These data not only demonstrate that NFAT5 in myeloid cells determines the Na+ content in BM, but that NFAT5 is able to govern the expression of the osteoprotective gene OPG. This provides insights into mechanisms of Na+-induced cessation of osteoclastogenesis and offers potentially new targets for treating salt-induced osteopenia.
Agnes Schröder, Patrick Neubert, Jens Titze, Aline Bozec, Wolfgang Neuhofer, Peter Proff, Christian Kirschneck, Jonathan Jantsch
Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone–associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.
Zhousheng Xiao, Gwendalyn King, Salvatore Mancarella, Undral Munkhsaikhan, Li Cao, Chun Cai, Leigh Darryl Quarles
The MEK1/2–ERK1/2 pathway has been implicated in regulating the inflammatory response to lung injury and infection, and pharmacologic MEK1/2 inhibitor compounds are reported to reduce detrimental inflammation in multiple animal models of disease, in part through modulation of leukocyte responses. However, the specific contribution of myeloid MEK1 in regulating acute lung injury (ALI) and its resolution remain unknown. Here, the role of myeloid Mek1 was investigated in a murine model of LPS-induced ALI (LPS-ALI) by genetic deletion using the Cre-floxed system (LysMCre × Mekfl), and human alveolar macrophages from healthy volunteers and patients with acute respiratory distress syndrome (ARDS) were obtained to assess activation of the MEK1/2–ERK1/2 pathway. Myeloid Mek1 deletion results in a failure to resolve LPS-ALI, and alveolar macrophages lacking MEK1 had increased activation of MEK2 and the downstream target ERK1/2 on day 4 of LPS-ALI. The clinical significance of these findings is supported by increased activation of the MEK1/2–ERK1/2 pathway in alveolar macrophages from patients with ARDS compared with alveolar macrophages from healthy volunteers. This study reveals a critical role for myeloid MEK1 in promoting resolution of LPS-ALI and controlling the duration of macrophage proinflammatory responses.
Matthew E. Long, Ke-Qin Gong, William E. Eddy, Joseph S. Volk, Eric D. Morrell, Carmen Mikacenic, T. Eoin West, Shawn J. Skerrett, Jean Charron, W. Conrad Liles, Anne M. Manicone
NK cells contribute to protective antitumor immunity, but little is known about the functional states of NK cells in human solid tumors. To address this issue, we performed single-cell RNA-seq analysis of NK cells isolated from human melanoma metastases, including lesions from patients who had progressed following checkpoint blockade. This analysis identified major differences in the transcriptional programs of tumor-infiltrating compared with circulating NK cells. Tumor-infiltrating NK cells represented 7 clusters with distinct gene expression programs indicative of significant functional specialization, including cytotoxicity and chemokine synthesis programs. In particular, NK cells from 3 clusters expressed high levels of XCL1 and XCL2, which encode 2 chemokines known to recruit XCR1+ cross-presenting DCs into tumors. In contrast, NK cells from 2 other clusters showed a higher level of expression of cytotoxicity genes. These data reveal key features of NK cells in human tumors and identify NK cell populations with specialized gene expression programs.
Lucas Ferrari de Andrade, Yuheng Lu, Adrienne Luoma, Yoshinaga Ito, Deng Pan, Jason W. Pyrdol, Charles H. Yoon, Guo-Cheng Yuan, Kai W. Wucherpfennig
Raeh Madhi, Milladur Rahman, Dler Taha, Johan Linders, Mohammed Merza, Yongzhi Wang, Matthias Mörgelon, Henrik Thorlacius
Toll-like receptor 3 (TLR3) is a pathogen recognition molecule associated with viral infection with double-stranded RNA (dsRNA) as its ligand. We evaluated the role of TLR3 in bacterial pneumonia using Klebsiella pneumoniae (KP). WT and TLR3–/– mice were subjected to a lethal model of KP. Alveolar macrophage polarization, bactericidal activity, and phagocytic capacity were compared. RNA-sequencing was performed on alveolar macrophages from the WT and TLR3–/– mice. Adoptive transfers of alveolar macrophages from TLR3–/– mice to WT mice with KP were evaluated for survival. Expression of TLR3 in postmortem human lung samples from patients who died from gram-negative pneumonia and pathological grading of pneumonitis was determined. Mortality was significantly lower in TLR3–/–, and survival improved in WT mice following antibody neutralization of TLR3 and with TLR3/dsRNA complex inhibitor. Alveolar macrophages from TLR3–/– mice demonstrated increased bactericidal and phagocytic capacity. RNA-sequencing showed an increased production of chemokines in TLR3–/– mice. Adoptive transfer of alveolar macrophages from the TLR3–/– mice restored the survival in WT mice. Human lung samples demonstrated a good correlation between the grade of pneumonitis and TLR3 expression. These data represent a paradigm shift in understanding the mechanistic role of TLR3 in bacterial pneumonia.
Madathilparambil V. Suresh, Vladislav A. Dolgachev, Boya Zhang, Sanjay Balijepalli, Samantha Swamy, Jashitha Mooliyil, Georgia Kralovich, Bivin Thomas, David Machado-Aranda, Monita Karmakar, Sanjeev Lalwani, Arulselvi Subramanian, Arun Anantharam, Bethany B. Moore, Krishnan Raghavendran
Small molecule inhibitors of Dual Specificity, Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A), including harmine and others, are able to drive human beta cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only, or the most important target, is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human beta cell RNAseq, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B; simultaneous silencing of both DYRK1A and DYRK1B yields greater beta cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to seven targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human beta cell proliferation, and provide clarity for future efforts at structure-based drug design for human beta cell regenerative drugs.
Courtney Ackeifi, Ethan Swartz, Kunal Kumar, Hongtao Liu, Suebsuwong Chalada, Esra Karakose, Donald K. Scott, Adolfo Garcia-Ocaña, Roberto Sanchez, Robert J. DeVita, Andrew F. Stewart, Peng Wang
Subpial demyelination is a specific hallmark of multiple sclerosis (MS) and a correlate of disease progression. Although the mechanism(s) that mediate pathogenesis in the subpial compartment remain unclear, it has been speculated that inflammation in the overlying meninges may be associated with subpial injury. Here we show that adoptive transfer of proteolipid protein-primed Th17 cells into SJL/J recipient mice induces subpial demyelination associated with microglial/macrophage activation, disruption of the glial limitans and evidence of an oxidative stress response. This pathology was topologically associated with foci of immune cells in the meninges and occurred in the absence of measurable anti-MOG IgM or IgG antibodies. To test the role of brain-infiltrating leukocytes on subpial injury, we modulated sphingosine 1-phosphate (S1P) receptor1,5 activity with BAF312 (siponimod) treatment. Administration of BAF312, even after adoptively transferred T cells had entered the brain, significantly ameliorated clinical EAE and diminished subpial pathology, concomitant with a selective reduction in the capacity of transferred T cells to make Th17 cytokines. We conclude that sustained subpial cortical injury is associated with the capacity for brain-resident T cells to produce Th17 cytokines, and this pathological process occurs in an S1P receptor1,5-dependent manner.
Lesley A. Ward, Dennis S.W. Lee, Anshu Sharma, Angela Wang, Ikbel Naouar, Xianjie I. Ma, Natalia Pikor, Barbara Nuesslein-Hildesheim, Valeria Ramaglia, Jennifer L. Gommerman
BACKGROUND We hypothesized that obesity-associated hepato-steatosis served as a pathophysiologic chemical depot for fat-soluble vitamins and altered normal physiology. Using α-tocopherol (vitamin E) as a model vitamin, pharmacokinetics and kinetics principles were utilized to determine whether excess liver fat sequestered α-tocopherol in women with obesity-associated hepato-steatosis vs healthy controls. METHODS Custom-synthesized deuterated α-tocopherols (d3- and d6-α-tocopherols) were administered to hospitalized healthy women and women with hepato-steatosis under IND guidelines. Serial samples obtained over 72 hours were analyzed by LC/MS. Fluorescent-labelled α-tocopherol was custom-synthesized for cell studies. RESULTS In healthy subjects, 85% of intravenous d6-α-tocopherol disappeared from the circulation within 20 minutes but reappeared within minutes and peaked at 6-8 hours. d3- and d6-α-Tocopherols localized to lipoproteins. Lipoprotein redistribution occurred only in vivo within 1h, indicating a key role of liver in rapid uptake and re-release into the circulation. Compared to healthy subjects, subjects with hepato-steatosis had similar d6-α-tocopherol entry rates into liver, but reduced initial release rates (p<0.001). Similarly, pharmacokinetics parameters of AUC and Maximum Concentration (Cmax) were reduced (AUC0-8 ,p<0.01;Cmax p<0.02) in hepato-steatosis subjects, indicating reduced hepatic d6-α-tocopherol output. Reduced kinetics and pharmacokinetics parameters (AUC and Cmax) in hepato-steatosis subjects who received 2 mg were mirrored by similar reductions in healthy subjects when comparing 5 and 2 mg doses. In vitro, fluorescent-labelled α-tocopherol localized specifically to lipid in fat-loaded hepatocytes, indicating sequestration. CONCLUSIONS The unique role of the liver in vitamin E physiology is dysregulated by excess liver fat. Obesity-associated hepato-steatosis may produce unrecognized hepatic vitamin E sequestration, which might subsequently drive liver disease. Our findings raise the possibility that hepato-steatosis may similarly alter hepatic physiology of other fat-soluble vitamins.
Pierre-Christian Violet, Ifechukwude C. Ebenuwa, Yu Wang, Mahtab Niyyati, Sebastian J. Padayatty, Brian Head, Kenneth Wilkins, Stacey Chung, Varsha Thakur, Lynn Ulatowski, Jeffrey Atkinson, Mikel Ghelfi, Sheila Smith, Hongbin Tu, Gerd Bobe, Chia-Ying Liu, David W. Herion, Robert D. Shamburek, Danny Manor, Maret G. Traber, Mark Levine
Overexpression and long terminal repeat (LTR) polymorphism of the HRES-1/Rab4 human endogenous retrovirus locus have been associated with T-cell activation and disease manifestations in systemic lupus erythematosus (SLE). Although genomic DNA methylation is overall diminished in SLE, its role in HRES-1/Rab4 expression is unknown. Therefore, we determined how lupus-associated polymorphic rs451401 alleles of the LTR regulate transcription from the HRES-1/Rab4 promoter and thus impact T-cell activation. The results show that cytosine-119 is hypermethylated while cytosine-51 of the promoter and the LTR-enhancer are hypomethylated in SLE. Pharmacological or genetic inactivation of DNA-methyltransferase-1 augmented the expression of HRES-1/Rab4. The minimal promoter was selectively recognized by metabolic stress sensor NRF1 when cytosine-119 but not cytosine-51 was methylated, and NRF1 stimulated HRES-1/Rab4 expression in human T cells. In turn, IRF2 and PSIP1 bound to the LTR-enhancer and exerted control over HRES-1/Rab4 expression in rs451401-genotype and methylation-dependent manners. The LTR-enhancer conferred markedly greater expression of HRES-1/Rab4 in subjects with rs451401CC over rs451401GG alleles that in turn promoted mechanistic target of rapamycin (mTOR) activation upon T-cell receptor stimulation. HRES-1/Rab4 alone robustly activated mTOR in human T cells. These findings identify HRES-1/Rab4 as a methylation and rs451401 allele-dependent transducer of environmental stress and controller of T-cell activation.
Aparna Godavarthy, Ryan Kelly, John Jimah, Miguel Beckford, Tiffany Caza, David Fernandez, Nick Huang, Manuel Duarte, Joshua Lewis, Hind J. Fadel, Eric M. Poeschla, Katalin Banki, Andras Perl
Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption – due to a complete absence of enteroendocrine cells – followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic, and hence may have had residual function in pancreas. We report two patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy, but instead developed permanent IDDM during middle childhood ages. The variants show no evidence of function in traditional promoter-based assays of NEUROG3 function and also fail to exhibit function in a variety of novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression, and hence suggests the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding beta-cell mass sufficient to maintain euglycemia until early childhood.
R. Sergio Solorzano-Vargas, Matthew Bjerknes, Jiafang Wang, S. Vincent Wu, Manuel G. Garcia-Careaga, Duke Pisit, Hazel Cheng, Michael S. German, Senta Georgia, Martin G. Martín
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