Inherited bone marrow failure syndromes (IBMFSs) such as Fanconi Anemia (FA) and Shwachman-Diamond syndrome (SDS) feature progressive cytopenia and a risk of acute myeloid leukemia (AML). Using deep phenotypic analysis of early progenitors in FA/SDS bone marrow samples we revealed selective survival of progenitors that phenotypically resembled granulocyte-monocyte progenitors (GMP). Whole exome and targeted sequencing of GMP-like cells in leukemia-free patients revealed a higher mutation load than in healthy controls and molecular changes that are characteristic of AML: increased G>A/C>T variants, decreased A>G/T>C variants, increased trinucleotide mutations at Xp(C>T)pT and decreased mutation rates at Xp(C>T)pG sites compared to other Xp(C>T)pX sites and enrichment for Cancer signature 1 (X indicates any nucleotide). Potential pre-leukemic targets in the GMP-like cells from FA/SDS patients included SYNE1, DST, HUWE1, LRP2, NOTCH2 and TP53. Serial analysis of GMPs from a SDS patient, who progressed to leukemia revealed a gradual increase in mutational burden, enrichment of G>A/C>T signature and emergence of new clones. Interestingly, the molecular signature of marrow cells from two FA/SDS patients with leukemia was similar to that of FA/SDS patients without transformation. The predicted founding clones in SDS-AML harbored mutations in several genes including TP53, while in FA-AML the mutated genes included ARID1B and SFPQ. We described an architectural change in the hematopoietic hierarchy of FA/SDS with remarkable preservation of GMP-like populations harboring unique mutation signatures. GMP-like cells might represent a cellular reservoir for clonal evolution.
Stephanie Claudia Heidemann, Brian Bursic, Sasan Zandi, Hongbing Li, Sagi Abelson, Robert J. Klaassen, Sharon Abish, Meera S Rayar, Vicky R. Breakey, Houtan Moshiri, Santhosh Dhanraj, Richard de Borja, Adam Shlien, John E. Dick, Yigal Dror
Background: Liver disease in urea cycle disorders (UCDs) ranges from hepatomegaly and chronic hepatocellular injury to cirrhosis and end-stage liver disease. However, the prevalence and underlying mechanisms are unclear. Methods: We estimated the prevalence of chronic hepatocellular injury in UCDs using data from a multicenter, longitudinal, natural history study. We also used ultrasound with shear wave elastography and FibroTestTM to evaluate liver stiffness and markers of fibrosis in individuals with argininosuccinate lyase deficiency (ASLD), a disorder with high prevalence of elevated serum alanine aminotransferase (ALT). To understand the human observations, we evaluated the hepatic phenotype of the AslNeo/Neo mouse model of ASLD. Results: We demonstrate a high prevalence of elevated ALT in ASLD (37%). Hyperammonemia and use of nitrogen-scavenging agents, two markers of disease severity, were significantly (p<0.001; p=0.001) associated with elevated ALT in ASLD. In addition, ultrasound with shear wave elastography and FibroTestTM revealed increased echogenicity and liver stiffness even in individuals with ASLD and normal aminotransferases. The AslNeo/Neo mice mimic the human disorder with hepatomegaly, elevated aminotransferases, and excessive hepatic glycogen noted prior to death (3-5 weeks of age). This excessive hepatic glycogen is associated with impaired hepatic glycogenolysis and decreased glycogen phosphorylase and is rescued with helper-dependent adenovirus expressing Asl using a liver specific (ApoE) promoter. Conclusions: Our results link urea cycle dysfunction and impaired hepatic glucose metabolism and identify a mouse model of liver disease in the setting of urea cycle dysfunction. Trial Registration: NCT03721367, NCT00237315 Funding: NIH, Burroughs Wellcome Fund, NUCDF, Genzyme/ACMG Foundation, CPRIT
Lindsay C. Burrage, Simran Madan, Xiaohui Li, Saima Ali, Mahmoud A. Mohammad, Bridget M. Stroup, Ming-Ming Jiang, Racel Cela, Terry Bertin, Jian Dai, Danielle Guffey, Milton Finegold, Sandesh Nagamani, Charles G. Minard, Juan Marini, Prakash Masand, Deborah Schady, Benjamin L. Shneider, Daniel H. Leung, Deeksha Bali, Brendan Lee
Group-2 innate lymphoid cells (ILC2s) are a critical innate source of type-2 cytokines in allergic inflammation. Although ILC2s are recognized as a critical cell population in the allergic inflammation, the regulatory mechanism(s) of ILC2s are less well understood. Here, we show that Regnase-1, an immune-regulatory RNase that degrades inflammatory mRNAs, negatively regulates ILC2 function, and that IkB kinase (IKK) complex-mediated Regnase-1 degradation is essential for IL-33– and IL-25–induced ILC2 activation. ILC2s from Regnase-1AA/AA mice expressing a Regnase-1 S435A/S439A mutant resistant to IKK complex-mediated degradation, accumulated Regnase-1 protein in response to IL-33 and IL-25. IL-33– and IL-25–stimulated Regnase-1AA/AA ILC2s showed reduced cell proliferation and type-2 cytokine (IL-5, IL-9, and IL-13) production and increased cell death. In addition, Il2ra and Il1rl1, but not Il5, Il9, or Il13, mRNAs were destabilized in IL-33–stimulated Regnase-1AA/AA ILC2s. In vivo, Regnase-1AA/AA mice showed attenuated acute type-2 pulmonary inflammation induced by the instillation of IL-33, IL-25, or papain. Furthermore, the expulsion of Nippostrongylus brasiliensis was significantly delayed in Regnase-1AA/AA mice. These results demonstrate that IKK complex-mediated Regnase-1 degradation is essential for ILC2s-mediated type-2 responses both in vitro and in vivo. Therefore, controlling Regnase-1 degradation is a potential therapeutic target for ILC2-contributed allergic disorders.
Kazufumi Matsushita, Hiroki Tanaka, Koubun Yasuda, Takumi Adachi, Ayumi Fukuoka, Shoko Akasaki, Atsuhide Koida, Etsushi Kuroda, Shizuo Akira, Tomohiro Yoshimoto
Lesch–Nyhan disease (LND) is a rare monogenic disease caused by deficiency of the salvage pathway enzyme hypoxanthine–guanine phosphoribosyltransferase (HGPRT) and is characterized by severe neuropsychiatric symptoms that currently cannot be treated. Predictive in vivo models are lacking for screening and evaluating candidate drugs because LND-associated neurological symptoms are not recapitulated in HGPRT-deficient animals. Here, we used human neural stem cells and neurons derived from induced pluripotent stem cells (iPSCs) of children affected by LND to identify neural phenotypes of interest associated with HGPRT deficiency to develop a target-agnostic-based drug screening system. We screened more than 3000 molecules and identified 6 pharmacological compounds, all possessing an adenosine moiety, that corrected HGPRT deficiency-associated neuronal phenotypes by promoting metabolism compensations in an HGPRT-independent manner. This included S-adenosylmethionine (SAM), a compound that had already been used as a compassionate approach to ease the neuropsychiatric symptoms in LND. Interestingly, these compounds compensate abnormal metabolism in a manner complementary to the gold standard allopurinol and can be provided to LND patients via simple food supplementation. This experimental paradigm can be easily adapted to other metabolic disorders affecting normal brain development and functioning in the absence of a relevant animal model.
Valentin Ruillier, Johana Tournois, Claire Boissart, Marie Lasbareilles, Gurvan Mahé, Laure Chatrousse, Michel Cailleret, Marc Peschanski, Alexandra Benchoua
Interleukin-3 (IL3) receptor α (IL3Rα) is the alpha subunit of the ligand-specific IL3 receptor and initiates intracellular signaling in response to IL3. IL3 amplifies pro-inflammatory signaling and cytokine storm in murine sepsis models. Here we found that RNFT2 (RING finger transmembrane-domain containing protein 2, also TMEM118), a previously uncharacterized RING finger ubiquitin E3 ligase, negatively regulated IL3-dependent cellular responses through IL3Rα ubiquitination and degradation in the proteasome. In vitro, IL3 stimulation promoted IL3Rα proteasomal degradation dependent on RNFT2, and we identified IL3Rα Lysine 357 as a ubiquitin acceptor site. We determined that LPS-priming reduces RNFT2 abundance, extends IL3Rα half-life, and sensitizes cells to the effects of IL3, acting synergistically to increase pro-inflammatory signaling. In vivo, IL3 synergized with LPS to exacerbate lung inflammation in LPS and Pseudomonas aeruginosa-challenged mice; conversely, IL3 neutralization reduced LPS-induce lung injury. Further, RNFT2 over-expression reduced lung inflammation and injury, whereas Rnft2 knockdown exacerbated inflammatory responses in LPS-induced murine lung injury. Lastly, we examined RNFT2 and IL3Rα in human lung explants from patients with Cystic Fibrosis, and also showed that IL3 is elevated in mechanically-ventilated critically ill humans at risk for Acute Respiratory Distress Syndrome (ARDS). These results identify RNFT2 as a negative regulator of IL3Rα, and show a potential role for the RNFT2/IL3Rα/IL3 axis in regulating innate immune responses in the lung.
Yao Tong, Travis B. Lear, John Evankovich, Yanwen Chen, James D. Londino, Michael M. Myerburg, Yingze Zhang, Iulia D. Popescu, John F. McDyer, Bryan J. McVerry, Karina C. Lockwood, Michael J. Jurczak, Yuan Liu, Bill B. Chen
Community-acquired pneumonia is a widespread disease with significant morbidity and mortality. Alveolar macrophages are tissue-resident lung cells that play a crucial role in innate immunity against bacteria that cause pneumonia. We hypothesized that alveolar macrophages display adaptive characteristics after resolution of bacterial pneumonia. We studied mice one to six months after self-limiting lung infections with Streptococcus pneumoniae, the most common cause of bacterial pneumonia. Alveolar macrophages, but not other myeloid cells recovered from the lung, showed long-term modifications of their surface marker phenotype. The remodeling of alveolar macrophages was: (i) long-lasting (still observed 6 months post infection), (ii) regionally localized (only observed in the affected lobe after lobar pneumonia), and (iii) associated with macrophage-dependent enhanced protection against another pneumococcal serotype. Metabolomic and transcriptomic profiling revealed that alveolar macrophages of mice that recovered from pneumonia had new baseline activities and altered responses to infection that better resembled those of adult humans. The enhanced lung protection after mild and self-limiting bacterial respiratory infections includes a profound remodeling of the alveolar macrophage pool that is long-lasting, compartmentalized, and manifest across surface receptors, metabolites, and both resting and stimulated transcriptomes.
Antoine Guillon, Emad I. Arafa, Kimberly A. Barker, Anna C. Belkina, Ian M.C. Martin, Anukul T. Shenoy, Alicia K. Wooten, Carolina Lyon De Ana, Anqi Dai, Adam Labadorf, Jaileene Hernandez-Escalante, Hans Dooms, Helene Blasco, Katrina E. Traber, Matthew R. Jones, Lee J. Quinton, Joseph P. Mizgerd
Alcohol abuse is a major public health problem worldwide causing a wide range of preventable morbidity and mortality. In this translational study, we show that heavy drinking (HD) (≥6 standard drinks/day) is independently associated to a worse outcome of ischemic stroke patients. To study the underlying mechanisms of this deleterious effect of HD, we then performed an extensive analysis of the brain inflammatory responses of mice exposed or not to 10% alcohol before and after ischemic stroke. Inflammatory responses were analyzed at the parenchymal, perivascular and vascular levels by using transcriptomic, immunohistochemical, in vivo two-photon microscopy and molecular MRI analyses. Alcohol-exposed mice show, in the absence of any other insult, a neurovascular inflammatory priming [i.e., an abnormal inflammatory status including an increase in brain perivascular macrophages (PVM)] associated to exacerbated inflammatory responses after a secondary insult (ischemic stroke or LPS challenge). Similar to our clinical data, alcohol-exposed mice showed larger ischemic lesions. We show here that PVM are key players on this aggravating effect of alcohol, since their specific depletion blocks the alcohol-induced aggravation of ischemic lesions. This study opens new therapeutic avenues aiming at blocking alcohol-induced exacerbation of the neurovascular inflammatory responses triggered after ischemic stroke.
Antoine Drieu, Anastasia Lanquetin, Damien Levard, Martina Glavan, Francisco Campos, Aurélien Quenault, Eloïse Lemarchand, Mikael Naveau, Anne Lise Pitel, José Castillo, Denis Vivien, Marina Rubio
ADPKD is the most common genetic cause of end stage kidney disease (ESRD). The treatment options for ADPKD are limited. We observed an upregulation in several IGF-1 pathway genes in the kidney of the Pkd1RC/RC mice. Pregnancy-associated plasma protein-A (PAPP-A), a metalloproteinase which cleaves inhibitory insulin-like growth factor binding proteins (IGFBPs), increasing the local bioactivity of IGF-1 was highly induced in the kideny of ADPKD mice. PAPP-A levels were high in cystic fluid and kidneys of humans with ADPKD. Our studies further showed the PAPP-A transcription in ADPKD is mainly regulated through the cAMP/CREB/CBP/p300 pathways. Pappa deficiency effectively inhibited the development of cysts in Pkd1RC/RC model of ADPKD. The role of PAPP-A in cystic disease appears to be regulation of the IGF-1 pathway and cellular proliferation in the kidney. Finally, preclinical studies demonstrated that treatment with monoclonal antibody that blocks the proteolytic activity of PAPP-A against IGFBP4 ameliorated ADPKD cystic disease in vivo in Pkd1RC/RC mice and ex vivo in embryonic kidneys. These data clearly indicated that the PAPP-A/IGF-1 pathway plays an important role in the growth and expansion of cysts in ADPKD. Our findings introduce a new therapeutic strategy for ADPKD that is the inhibition of PAPP-A.
Sonu Kashyap, Kyaw Zaw Hein, Claudia C.S. Chini, Jorgo Lika, Gina M. Warner, Laurie K. Bale, Vicente E. Torres, Peter C. Harris, Claus Oxvig, Cheryl A. Conover, Eduardo N. Chini
Adequate iron supply during pregnancy is essential for fetal development. However, how fetal or amniotic fluid iron levels are regulated during healthy pregnancy, or pregnancies complicated by intraamniotic infection or inflammation (IAI) is unknown. We evaluated amniotic fluid and fetal iron homeostasis in normal and complicated murine, macaque, and human pregnancy. In mice, fetal iron endowment was affected by maternal iron status but amniotic fluid iron concentrations changed little during maternal iron deficiency or excess. In murine and macaque models of inflamed pregnancy, the fetus responded to maternal systemic inflammation or IAI by rapidly upregulating hepcidin and lowering iron in fetal blood, without altering amniotic fluid iron. In humans, elevated cord blood hepcidin with accompanying hypoferremia was observed in pregnancies with antenatal exposure to IAI compared to those that were non-exposed. Hepcidin was also elevated in human amniotic fluid from pregnancies with IAI compared to those without IAI, but amniotic fluid iron levels did not differ between the groups. Our studies in mice, macaques, and humans demonstrate that amniotic fluid iron is largely unregulated but that the rapid induction of fetal hepcidin by inflammation and consequent fetal hypoferremia are conserved mechanisms that may be important in fetal host defense.
Allison L. Fisher, Veena Sangkhae, Pietro Presicce, Claire A. Chougnet, Alan H. Jobe, Suhas G. Kallapur, Sammy M. Tabbah, Catalin S. Buhimschi, Irina A. Buhimschi, Tomas Ganz, Elizabeta Nemeth
Systemic lupus erythematosus (SLE) is a complex autoimmune disease that follows an unpredictable disease course and affects multiple organs and tissues. We performed an integrated, multi-cohort analysis of 7,471 transcriptomic profiles from 40 independent studies to identify robust gene expression changes associated with SLE. We identified a 93-gene signature (SLE MetaSignature) that is differentially expressed in the blood of SLE patients compared to healthy volunteers; distinguishes SLE from other autoimmune, inflammatory, and infectious diseases; and persists across diverse tissues and cell types. The SLE MetaSignature correlated significantly with disease activity and other clinical measures of inflammation. We prospectively validated the SLE MetaSignature in an independent cohort of pediatric SLE patients using a microfluidic RT-qPCR array. We found that 14 of the 93 genes in the SLE MetaSignature were independent of interferon-induced and neutrophil-related transcriptional profiles that have previously been associated with SLE. Pathway analysis revealed dysregulation associated with nucleic acid biosynthesis and immunometabolism in SLE. We further refined a neutropoeisis signature and identified novel transcripts related to immune cells and oxidative stress. Our multi-cohort, transcriptomic analysis has uncovered novel genes and pathways associated with SLE pathogenesis, with the potential to advance clinical diagnosis, biomarker development, and targeted therapeutics for SLE.
Winston A. Haynes, David James Haddon, Vivian Diep, Avani Khatri, Erika Bongen, Gloria Yiu, Imelda Balboni, Christopher R. Bolen, Rong Mao, Paul J. Utz, Purvesh Khatri
Pulmonary Langerhans cell histiocytosis (PLCH) is a rare, smoking-related, lung disease characterized by dendritic cell (DC) accumulation, bronchiolocentric nodule formation, and cystic lung remodeling. Approximately 50% of PLCH patients harbor somatic BRAF-V600E mutations in cells of the myeloid/monocyte lineage. However, the rarity of the disease and lack of animal models has impeded the study of PLCH pathogenesis. Here, we established a cigarette smoke (CS)-exposed, BRAF-V600E mutant mouse model that recapitulates many hallmark characteristics of PLCH. We show that CD11c-targeted expression of BRAF-V600E increases DC responsiveness to stimuli, including the chemokine CCL20, and that mutant DC accumulation in the lungs of CS-exposed mice is due to both increased cellular viability and enhanced recruitment. Moreover, we report that the chemokine CCL7 is secreted from DCs and human peripheral blood monocytes in a BRAF-V600E-dependent manner, suggesting a possible mechanism for recruitment of cells known to dominate PLCH lesions. Inflammatory lesions and airspace dilation in BRAF-V600E mice in response to CS are attenuated by transitioning animals to filtered air and treatment with a BRAF-V600E inhibitor, PLX4720. Collectively, this model provides mechanistic insights into the role of DCs, the BRAF-V600E mutation and CS exposure in PLCH pathogenesis, and provides a platform to develop therapeutic targets.
Huan Liu, Andrew R. Osterburg, Jennifer Flury, Zulma Swank, Dennis W. McGraw, Nishant Gupta, Kathryn A. Wikenheiser-Brokamp, Ashish Kumar, Abdellatif Tazi, Yoshikazu Inoue, Masaki Hirose, Francis X. McCormack, Michael Borchers
Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase-mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2f/f) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2f/f). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting co-factors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell-extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.
Steven Tessier, Alexandra C Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud
Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (> 95%) of myocardial B cells remain intravascular, whereas few (< 5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.
Luigi Adamo, Cibele Rocha-Resende, Chieh-Yu Lin, Sarah Evans, Jesse W. Williams, Hao Dun, Wenjun Li, Cedric Mpoy, Prabhakar Andhey, Buck E. Rogers, Kory Lavine, Daniel Kreisel, Maxim N. Artyomov, Gwendalyn J. Randolph, Douglas Mann
OBJECTIVES: Idiopathic inflammatory myopathies (IIM) are characterized by muscle inflammation and weakness, myositis specific autoantibodies (MSAs) and extramuscular organ damage. The role of neutrophil dysregulation and neutrophil extracellular traps (NETs) in IIM is unclear. We assessed if pathogenic neutrophil subsets (low-density granulocytes, LDGs) and NETs were elevated in IIM, associated with clinical presentation and MSAs, and their effect on skeletal myoblasts and myotubes.METHODS: Circulating NETs and LDGs were quantified and correlated with clinical measures. Specific MSAs were tested for their ability to induce NETs. NETs and neutrophil gene expression were measured in IIM biopsies. Whether NETs damage skeletal myoblasts and myotubes was tested.RESULTS: Circulating LDGs and NETs were increased in IIM. IIM LDGs had enhanced ability to form NETs. LDGs and NETs correlated with IIM disease activity and muscle damage. The serum MSA anti-MDA5 correlated with circulating and tissue NETs and directly enhanced NET formation. An enhanced neutrophil gene signature was present in IIM muscle and associated with muscle injury and tissue interferon gene signatures. IIM NETs decreased the viability of myotubes in a citrullinated histone-dependent manner. CONCLUSION: Dysregulated neutrophil pathways may play pathogenic roles in IIM through their ability to directly injure muscle cells and other affected tissues.
Nickie L. Seto, Jose Jiram Torres-Ruiz, Carmelo Carmona-Rivera, Iago Pinal-Fernandez, Katherine Pak, Monica M. Purmalek, Yuji Hosono, Catia Fernandes-Cerqueira, Prateek C. Gowda, Nathan Arnett, Alexander Gorbach, Olivier Benveniste, Diana Gómez-Martín, Albert Selva-O'Callaghan, Jose C. Milisenda, Josep M. Grau-Junyent, Lisa Christopher-Stine, Frederick W. Miller, Ingrid E. Lundberg, J. Michelle Kahlenberg, Adam I. Schiffenbauer, Andrew L. Mammen, Lisa G. Rider, Mariana J. Kaplan
BACKGROUND. Siponimod (BAF312) is a selective sphingosine 1-phosphate receptor 1 and 5 (S1PR1, S1PR5) modulator recently approved for active secondary progressive multiple sclerosis (SPMS). The immunomodulatory effects of siponimod in SPMS have not been previously described. METHODS. We conducted a multi-centered randomized, double-blind, placebo-controlled AMS04 mechanistic study with 36 SPMS participants enrolled in the EXPAND trial. Gene expression profiles were analyzed using RNA derived from whole blood with Affymetrix Human Gene ST 2.1 microarray technology. We performed flow cytometry based assays to analyze the immune cell composition and microarray gene expression analysis on peripheral blood from siponimod-treated participants with SPMS relative to baseline and placebo during the first year randomization phase. RESULTS. Microarray analysis showed that immune-associated genes involved in T and B cell activation and receptor signaling were largely decreased by siponimod, which is consistent with the reduction of CD4+ T cells, CD8+ T cells, and B cells. Analysis done by flow cytometry showed that within the remaining lymphocyte subsets, there was a reduction in the frequencies of CD4 and CD8 naïve T cells and central memory cells, while T effector memory cells, anti-inflammatory Th2, and T regulatory (Treg) cells were enriched. Transitional Bregs (CD24hiCD38hi) and B1 cell subsets (CD43+CD27+) were enriched, shifting the balance in favor of regulatory B cells over memory B cells. The pro-regulatory shift driven by siponimod treatment included a higher proliferative potential of Tregs compared with non-Tregs, and upregulated expression of PD-1 on Tregs. Additionally, a positive correlation was found between regulatory T cells and regulatory B cells in siponimod treated participants. CONCLUSION. The shift toward an anti-inflammatory and suppressive homeostatic immune system may contribute to the clinical efficacy of siponimod in SPMS. TRIAL REGISTRATION. NCT02330965.
Qi Wu, Elizabeth A. Mills, Qin Wang, Catherine A. Dowling, Caitlyn Fisher, Britany Kirch, Steven K. Lundy, David A. Fox, Yang Mao-Draayer
Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for six months after infection with WT measles virus (MeV). Infection caused viremia and rash with clearance of infectious virus by 14 days. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H) and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral Tfh cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody to both H and N, with more H-specific ASCs in BM. From 14-21 days 20-100-fold more total ASCs than MeV-specific ASCs appeared in circulation suggesting mobilization of pre-existing ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.
Ashley N. Nelson, Wen-Hsuan W. Lin, Rupak Shivakoti, Nicole E. Putnam, Lisa M. Mangus, Robert J. Adams, Debra Hauer, Victoria K. Baxter, Diane E. Griffin
Alternative polyadenylation (APA) is a widespread and important mechanism in regulation of gene expression. Dysregulation of the 3’ UTR cleavage and polyadenylation represents a common characteristic among many disease states including lung fibrosis. In this study, we investigated the role of mammalian cleavage factor I (CFIm)-mediated APA in regulating the extracellular matrix production in response to mechanical stimuli from stiffened matrix simulating the fibrotic lungs. We found that stiff matrix downregulates expression of CFIm68, CFIm59 and CFIm25 subunits, and promotes APA in favor of the proximal poly(A) site usage in the 3’ UTRs of type I collagen (COL1A1) and fibronectin (FN1) in primary human lung fibroblasts. Knockdown and overexpression of each individual CFIm subunit demonstrated that CFIm68 and CFIm25 are indispensable attributes of stiff matrix-induced APA and overproduction of COL1A1, whereas CFIm does not appear to mediate stiffness-regulated FN1 APA. Furthermore, expression of the CFIm subunits is associated with matrix stiffness in vivo in a bleomycin-induced mouse model of pulmonary fibrosis. These data suggest that stiff matrix instigates type I collagen biogenesis by selectively targeting mRNA transcripts for 3’ UTR shortening. The current study uncovered a potential mechanism for regulation of the CFIm complex by mechanical cues under fibrotic conditions.
Zijing Zhou, Jing Qu, Li He, Yi Zhu, Shanzhong Yang, Feng Zhang, Ting Guo, Hong Peng, Ping Chen, Yong Zhou
Adult renal proximal tubules are composed of terminally differentiated epithelial cells that exhibit few signs of proliferation over time. However, upon acute kidney injury (AKI), surviving epithelial cells can re-enter the mitotic cycle and express genes and proteins coincident with a dedifferentiated, more embryonic phenotype. While a stable, terminally differentiated cellular phenotype is thought to be maintained, at least in part, by epigenetic imprints that impart both active and repressive histone marks, it is unclear whether regenerating cells after injury need to replicate such marks to recover. To test whether renal epithelial cell regeneration is dependent on histone H3K4 methylation, we generated a mouse model that deleted the Paxip1 gene in mature renal proximal tubules. Paxip1 encodes the adaptor protein PTIP, which is part of an Mll3/4 histone H3K4 methyltransferase complex and is essential for embryonic development. Mice with PTIP deletions in the adult kidney proximal tubules were viable and fertile. Upon acute kidney injury, such mice failed to regenerate damaged tubules leading to scarring and interstitial fibrosis. The inability to repair damage was likely due to a failure to re-enter mitosis and reactivate regulatory genes such as Sox9, which is necessary for epithelial cell regeneration. PTIP deletion reduced histone H3K4 methylation in uninjured adult kidneys but did not significantly affect function or the expression of epithelial specific markers. A transient decrease in trimethylation was also observed in controls after AKI but returned to normal after repair. Strikingly, cell lineage tracing revealed that surviving PTIP mutant cells could alter their phenotype and lose epithelial markers. These data demonstrate that PTIP and associated MLL3/4 mediated histone methylation are needed for regenerating proximal tubules and to maintain or reestablish the cellular epithelial phenotype.
Abdul Soofi, Ana P. Kutschat, Mohammad H. Azam, Ann M. Laszczyk, Gregory R. Dressler
Dengue (DENV) and Zika viruses (ZIKV) are closely related mosquito-borne flaviviruses that co-circulate in tropical regions and constitute major threats to global human health. Whether preexisting immunity to one virus affects disease caused by the other during primary or secondary infections is unknown but is critical in preparing for future outbreaks and predicting vaccine safety. Using a human skin explant model, we show that DENV-3 immune sera increased recruitment and infection of Langerhans cells, macrophages and dermal dendritic cells following inoculation with DENV-2 or ZIKV. Similarly, ZIKV immune sera enhanced infection with DENV-2. Immune sera increased migration of infected Langerhans cells to dermis and emigration of infected cells out of skin. Heterotypic immune sera increased viral RNA in dermis almost tenfold and reduced the amount of virus required to infect a majority of myeloid cells by 100 to 1,000 fold. Enhancement was associated with cross-reactive IgG and induction of IL-10 expression and was mediated by both CD32 and CD64 Fcγ receptors. These findings reveal that preexisting heterotypic immunity greatly enhances DENV and ZIKV infection, replication and spread in human skin. This relevant tissue model will be valuable in assessing the efficacy and risk of dengue and Zika vaccines in humans.
Priscila M.S. Castanha, Geza Erdos, Simon C. Watkins, Louis D. Falo, Jr., Ernesto T.A. Marques, Simon M. Barratt-Boyes
BACKGROUND. The circadian system entrains behavioral and physiological rhythms to environmental cycles and modern lifestyles disrupt this entrainment. We investigated a timed exercise intervention to phase shift the internal circadian rhythm. METHODS. In fifty-two young, sedentary adults, dim light melatonin onset (DLMO) was measured before and after five days of morning (10h after DLMO; n = 26) or evening (20h after DLMO; n = 26) exercise. Phase shifts were calculated as the difference in DLMO before and after exercise. RESULTS. Morning exercise induced phase advance shifts (0.62 ± 0.18h) that were significantly greater than phase shifts from evening exercise (-0.02 ± 0.18h; P = 0.01). Chronotype also influenced the effect of timed exercise. For later chronotypes, both morning and evening exercise induced phase advances (0.54 ± 0.29h and 0.46 ±0.25h, respectively). In contrast, earlier chronotypes had phase advances from morning exercise (0.49 ± 0.25h), but phase delays from evening exercise (-0.41 ± 0.29h). CONCLUSION. Late chronotypes, who experience the most severe circadian misalignment, may benefit from phase advances induced by exercise in the morning or evening, but evening exercise may exacerbate circadian misalignment in early chronotypes. Thus, personalized exercise timing prescription based on chronotype could alleviate circadian misalignment in young adults. TRIAL REGISTRATION. www.clinicaltrials.gov, NCT # NCT04097886.FUNDING. National Institutes of Health grants UL1TR001998 and TL1TR001997, the Barnstable Brown Diabetes and Obesity Center, the Pediatric Exercise Physiology Laboratory Endowment, the Arvle and Ellen Turner Thacker Research Fund, and the University of Kentucky.
J. Matthew Thomas, Philip A. Kern, Heather M. Bush, Kristen J. McQuerry, W. Scott Black, Jody L. Clasey, Julie S. Pendergast