Introduction: Granzyme B (GzmB) is a serine protease involved in immune cell-mediated apoptosis that is enabled through a mechanism involving the pore-forming protein, perforin that facilitates internalization. However, recent evidence suggests that GzmB contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. Hypothesis: GzmB contributes to cardiac fibrosis through a perforin-independent pathway involving extracellular proteolysis. Methods: Using a murine model of Angiotensin II (Ang II)-induced cardiac fibrosis, wild-type, GzmB deficient and Perforin deficient mice were treated with Ang II for 4 weeks, and were examined for the presence of cardiac fibrosis. Echocardiography was performed in these mice to examine the cardiac function. The level of Inflammation and inflammatory cells infiltration were examined by immunohistochemistry and RT-PCR analysis. The in vitro endothelial barrier function was measured by electric cell-substrate impedance sensing. Results: GzmB was highly up-regulated in both murine and human cardiac fibrosis. Genetic deficiency of GzmB markedly reduced Ang II-induced cardiac dysfunction, hypertrophy and fibrosis, independently of perforin. GzmB deficiency also decreases microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro studies identified VE-cadherin as a GzmB substrate. VE-cadherin is a key endothelial cell-cell junction protein. GzmB-mediated VE-cadherin cleavage resulted in increased endothelial permeability, and increased transcellular conductance. These results were also observed in vivo. Conclusions: GzmB contributes to the onset and progression of cardiac fibrosis through a perforin-independent process involving the cleavage of VE-cadherin.
ABSTRACT Discovery of humans with monogenic disorders has a rich history of generating new insights into biology. Here we report the first human identified with complete deficiency of nuclear factor of activated T cells 1 (NFAT1). NFAT1, encoded by NFATC2 , mediates calcium-calcineurin signals that drive cell activation, proliferation, and survival. The patient is homozygous for a damaging germline NFATC2 variant (c.2023_2026delTACC; p.Tyr675Thrfs*18) and presented with joint contractures, osteochondromas, and B cell lymphoma. Absence of NFAT1 protein in chondrocytes caused enrichment in pro-survival and inflammatory genes. Systematic single-cell-omic analyses revealed an environment that promotes lymphomagenesis with accumulation of naïve B cells (with oncogenic signatures - MYC , JAK1 ), exhausted CD4 + T cells, impaired T follicular helper cells, and aberrant CD8 + T cells. This work highlights the pleiotropic role of human NFAT1, will empower the diagnosis of additional patients with NFAT1 deficiency, and further define detrimental effects a long-term use of calcineurin inhibitors.
The innate immune system allows for rapid recognition of pathogens. Toll-like receptor (TLR) signaling is a key aspect of the innate immune response, and interleukin-1 receptor-associated kinase 4 (IRAK4) plays a vital role in the TLR signaling cascade. Each TLR recognizes a distinct set of pathogen-associated molecular patterns (PAMPs) that encompass conserved microbial components such as lipopolysaccharides and flagellin. Upon binding of PAMPs and TLR activation, TLR intracellular domains initiate the oligomerization of the myeloid differentiation primary response 88 (MyD88), IRAK1, IRAK2, and IRAK4 signaling platform known as the Myddosome complex while also triggering the Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent pathway. The Myddosome complex initiates signal transduction pathways enabling the activation of NF-κB and mitogen-activated protein kinase (MAPK) transcription factors and the subsequent production of inflammatory cytokines. Human IRAK4 deficiency is an autosomal recessive inborn error of immunity that classically presents with blunted or delayed inflammatory response to infection and susceptibility to a narrow spectrum of pyogenic bacteria, particularly Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. We describe a case of IRAK4 deficiency in an 11-mo-old boy with concurrent S. pneumoniae bacteremia and S. aureus cervical lymphadenitis with a blunted inflammatory response to invasive infection. Although initial clinical immune profiling was unremarkable, a high degree of suspicion for an innate immune defect prompted genetic sequencing. Genetic testing revealed a novel variant in the IRAK4 gene (c.1049delG, p.(Gly350Glufs*15)) predicted to be likely pathogenic. Functional testing showed a loss of IRAK4 protein expression and abolished TLR signaling, confirming the pathogenicity of this novel IRAK4 variant.
Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II–induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II–induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis. Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II–induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II–induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis. Cardiac fibrosis is a common pathological feature of many heart diseases, and a hallmark feature of chronic heart failure.1Berk B.C. Fujiwara K. Lehoux S. ECM remodeling in hypertensive heart disease.J Clin Invest. 2007; 117: 568-575Crossref PubMed Scopus (685) Google Scholar Heart failure affects more than five million individuals in North America, and costs >$32 billion annually in health care services, medications, and lost productivity.2Go A.S. Mozaffarian D. Roger V.L. Benjamin E.J. Berry J.D. Borden W.B. et al.American Heart Association Statistics Committee, Stroke Statistics SubcommitteeHeart disease and stroke statistics–2013 update: a report from the American Heart Association.Circulation. 2013; 127: e6-e245Crossref PubMed Scopus (4361) Google Scholar Approximately half of the patients diagnosed with heart failure die within 5 years, often as a direct consequence of reduced cardiac function.3Yancy C.W. Jessup M. Bozkurt B. Butler J. Casey Jr., D.E. Drazner M.H. Fonarow G.C. Geraci S.A. Horwich T. Januzzi J.L. Johnson M.R. Kasper E.K. Levy W.C. Masoudi F.A. McBride P.E. McMurray J.J. Mitchell J.E. Peterson P.N. Riegel B. Sam F. Stevenson L.W. Tang W.H. Tsai E.J. Wilkoff B.L. 2013 ACCF/AHA guideline for the management of heart failure: executive summary: a report of the American College of Cardiology Foundation/American Heart Association Task Force on practice guidelines.Circulation. 2013; 128: 1810-1852Abstract Full Text Full Text PDF PubMed Scopus (4682) Google Scholar Cardiac fibrosis involves exaggerated deposition and reduced degradation of extracellular matrix (ECM) proteins, which directly affects heart function.4Diez J. Mechanisms of cardiac fibrosis in hypertension.J Clin Hypertens (Greenwich). 2007; 9: 546-550Crossref PubMed Scopus (166) Google Scholar Fibrosis leads to elevated mechanical stiffness,5Chaturvedi R.R. Herron T. Simmons R. Shore D. Kumar P. Sethia B. Chua F. Vassiliadis E. Kentish J.C. Passive stiffness of myocardium from congenital heart disease and implications for diastole.Circulation. 2010; 121: 979-988Crossref PubMed Scopus (118) Google Scholar poor electrical signaling,6Spach M.S. Boineau J.P. Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias.Pacing Clin Electrophysiol. 1997; 20: 397-413Crossref PubMed Scopus (260) Google Scholar and reduced oxygen delivery,7Kai H. Mori T. Tokuda K. Takayama N. Tahara N. Takemiya K. Kudo H. Sugi Y. Fukui D. Yasukawa H. Kuwahara F. Imaizumi T. Pressure overload-induced transient oxidative stress mediates perivascular inflammation and cardiac fibrosis through angiotensin II.Hypertens Res. 2006; 29: 711-718Crossref PubMed Scopus (91) Google Scholar with subsequent reduced cardiac pumping capacity and abnormal heart rhythm. Given this knowledge, new methods or therapeutics that may prevent or treat cardiac fibrosis are needed. However, despite decades of intense research, effective therapies for cardiac fibrosis remain elusive. Many distinct triggers can contribute to the development of progressive fibrotic disease. Despite having different etiologies, the common trigger for most chronic fibrotic disorders is persistent inflammation, reflected in enhanced production of cytokines and growth factors, up-regulated expression of proteolytic enzymes, and elevated infiltration of inflammatory cells.8Wynn T.A. Cellular and molecular mechanisms of fibrosis.J Pathol. 2008; 214: 199-210Crossref PubMed Scopus (3061) Google Scholar Persistent inflammation stimulates fibroblasts to produce an excess of ECM proteins, which ultimately impairs normal myocardial architecture and function.8Wynn T.A. Cellular and molecular mechanisms of fibrosis.J Pathol. 2008; 214: 199-210Crossref PubMed Scopus (3061) Google Scholar, 9Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2088) Google Scholar Although granzyme B (GzmB) was discovered as both an intracellular and an extracellular serine protease,10Boivin W.A. Cooper D.M. Hiebert P.R. Granville D.J. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma.Lab Invest. 2009; 89: 1195-1220Crossref PubMed Scopus (177) Google Scholar until recently, this protease was primarily viewed as a mediator of immune cell–mediated apoptosis, through a mechanism involving the membrane-perforating molecule, perforin, which allows GzmB entry into the cytoplasm of the target cell.11Jenne D.E. Tschopp J. Granzymes, a family of serine proteases released from granules of cytolytic T lymphocytes upon T cell receptor stimulation.Immunol Rev. 1988; 103: 53-71Crossref PubMed Scopus (224) Google Scholar Early studies overlooked the potential role of extracellular GzmB, using perforin-deficient (Prf1−/−) mice to study the role of granzymes in disease with the assumption that perforin is necessary for granzyme internalization and apoptosis. The major shortcoming of this approach was that it ignored any possibility that granzymes could contribute to disease independent of perforin, through extracellular mechanisms.12Froelich C.J. Pardo J. Simon M.M. Granule-associated serine proteases: granzymes might not just be killer proteases.Trends Immunol. 2009; 30: 117-123Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar Over recent years, the traditional pathophysiological understanding of GzmB has been challenged because of increasing data showing that GzmB accumulates in the extracellular space of inflamed tissue, and is present and retains its activity in body fluids collected from patients with diseases associated with aging and chronic inflammation.10Boivin W.A. Cooper D.M. Hiebert P.R. Granville D.J. Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma.Lab Invest. 2009; 89: 1195-1220Crossref PubMed Scopus (177) Google Scholar, 13Hiebert P.R. Granville D.J. Granzyme B in injury, inflammation, and repair.Trends Mol Med. 2012; 18: 732-741Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar In the extracellular milieu, independent of perforin, GzmB cleaves ECM into fragments that are capable of promoting chemotaxis and increased inflammation.14Buzza M.S. Zamurs L. Sun J. Bird C.H. Smith A.I. Trapani J.A. Froelich C.J. Nice E.C. Bird P.I. Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin.J Biol Chem. 2005; 280: 23549-23558Crossref PubMed Scopus (199) Google Scholar, 15Froelich C.J. Zhang X. Turbov J. Hudig D. Winkler U. Hanna W.L. Human granzyme B degrades aggrecan proteoglycan in matrix synthesized by chondrocytes.J Immunol. 1993; 151: 7161-7171PubMed Google Scholar, 16Boivin W.A. Shackleford M. Vanden Hoek A. Zhao H. Hackett T.L. Knight D.A. Granville D.J. Granzyme B cleaves decorin, biglycan and soluble betaglycan, releasing active transforming growth factor-beta1.PLoS One. 2012; 7: e33163Crossref PubMed Scopus (79) Google Scholar GzmB also modulates cytokine activation through its extracellular proteolytic activity and contributes to persistent inflammation.13Hiebert P.R. Granville D.J. Granzyme B in injury, inflammation, and repair.Trends Mol Med. 2012; 18: 732-741Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar In addition, GzmB can induce vascular permeability through the proteolytic release of ECM-sequestered vascular endothelial growth factor, and promote immune cell transmigration through cleavage of the basement membrane.17Hendel A. Hsu I. Granville D.J. Granzyme B releases vascular endothelial growth factor from extracellular matrix and induces vascular permeability.Lab Invest. 2014; 94: 716-725Crossref PubMed Scopus (34) Google Scholar, 18Prakash M.D. Munoz M.A. Jain R. Tong P.L. Koskinen A. Regner M. Kleifeld O. Ho B. Olson M. Turner S.J. Mrass P. Weninger W. Bird P.I. Granzyme B promotes cytotoxic lymphocyte transmigration via basement membrane remodeling.Immunity. 2014; 41: 960-972Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar Although there is mounting evidence for the involvement of GzmB in cellular processes related to fibrosis, there has been no direct link established between GzmB and cardiac fibrosis. In the present study, using a well-established mouse model of angiotensin II (Ang II)–induced cardiac fibrosis,19Lijnen P.J. Petrov V.V. Fagard R.H. Induction of cardiac fibrosis by angiotensin II.Methods Find Exp Clin Pharmacol. 2000; 22: 709-723Crossref PubMed Scopus (69) Google Scholar, 20Hou J. Kato H. Cohen R.A. Chobanian A.V. Brecher P. Angiotensin II-induced cardiac fibrosis in the rat is increased by chronic inhibition of nitric oxide synthase.J Clin Invest. 1995; 96: 2469-2477Crossref PubMed Scopus (82) Google Scholar we demonstrated that GzmB is up-regulated in the fibrotic heart. GzmB deficiency protected against Ang II–induced cardiac hypertrophy and cardiac fibrosis, whereas perforin deficiency had no protective effect. GzmB directly cleaved VE-cadherin, a key endothelial cell-cell junction protein, and contributed to the disruption of endothelial barrier function and an increase in vascular permeability. GzmB deficiency attenuated cardiac microvascular permeability, cardiac inflammation, and fibroblast accumulation in Ang II–infused hearts, to result in less cardiac fibrosis. On the basis of these data, we propose a perforin-independent, extracellular role of GzmB in the pathogenesis of cardiac fibrosis. Targeting extracellular GzmB could be a potential therapeutic strategy to intervene in the progression of cardiac fibrosis. Tissue samples from cases of heart failure from different etiologies, including atherosclerotic coronary artery disease (n = 3), idiopathic dilated cardiomyopathy (n = 4), healed myocarditis (n = 2), sarcoidosis (n = 1), and aortic stenosis (n = 1) with established patterns of fibrosis, were identified and provided by the staff of the Cardiovascular Tissue Registry of St. Paul's Hospital and University of British Columbia (UBC) in accordance with their ethics protocols. The clinical characteristics of the patients are summarized in Table 1. The analysis of human samples in this study was approved by UBC/Providence Health Care Research Ethics Board (H14-01716).Table 1The Clinical Characteristics of Study PatientsStudy groupCase no.SexAge, yearsPrimary cardiac diagnosisHealthy heart1Male37Normal2Male213Male21Ischemic heart disease with prior infarction4Male63Atherosclerotic coronary artery disease with prior myocardial infarction5Female506Male61Dilated cardiomyopathy7Female44Idiopathic dilated cardiomyopathy8Male269Male6110Male52Other diseases11Male59Aortic stenosis12Male56Active sarcoidosis13Male48Healed myocarditis14Male67Healed myocarditis Open table in a new tab Male wild-type (WT; C57BL/6J), GzmB-deficient (Gzmb−/− with C57BL/6J background), and perforin-deficient (Prf1−/− with C57BL/6J background) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed at the Genetic Engineered Models facility at the UBC Centre for Heart Lung Innovation, St. Paul's Hospital. Animals were fed and watered ad libitum and maintained on a 12-hour light/dark cycle. All animal procedures were performed in accordance with the guidelines for animal experimentation approved by the Animal Care Committee of UBC (A14-0230). Cardiac fibrosis was induced by Ang II infusion, as previously described.19Lijnen P.J. Petrov V.V. Fagard R.H. Induction of cardiac fibrosis by angiotensin II.Methods Find Exp Clin Pharmacol. 2000; 22: 709-723Crossref PubMed Scopus (69) Google Scholar Briefly, mice aged 3 to 4 months received either a 4-week Ang II (Sigma-Aldrich, St. Louis, MO) infusion at 1000 ng/minute per kilogram or a saline infusion from a s.c. implanted 1004 model ALZET mini osmotic pump (DURECT Corp., Cupertino, CA). An osmotic pump was filled with the appropriate solution, primed at 37°C for 24 hours in saline, and surgically implanted s.c. posterior to the scapula of the mouse. During the implantation procedure, mice were anesthetized with gaseous anesthetic at a flow rate of 1.5 L/minute of oxygen with 1.5% to 2.5% of isoflurane delivered via a Baines system using a calibrated tabletop anesthetic machine, administered from a rodent nose cone. Postsurgical pain control consisted of a s.c. injection of buprenorphine. Mean blood pressure and resting heart rates were measured by tail cuff plethysmography using the CODA blood pressure system (Kent Scientific Corp., Torrington, CT) before treatment and every week during Ang II infusion. Echocardiography was performed under mild isoflurane sedation (0.75% to 1.25%) using a VisualSonics Vevo 2100 High-Resolution Imaging System with a 40-MHz frequency transducer (Fujifilm VisualSonics, Toronto, ON, Canada). Two-dimensional M-mode images of the left ventricle at the papillary muscle level were obtained from the left parasternal short axis view. Calculations were made according to the guidelines of the American Society of Echocardiography.21Lang R.M. Bierig M. Devereux R.B. Flachskampf F.A. Foster E. Pellikka P.A. Picard M.H. Roman M.J. Seward J. Shanewise J.S. Solomon S.D. Spencer K.T. Sutton M.S. Stewart W.J. Chamber Quantification Writing Group, American Society of Echocardiography's Guidelines and Standards Committee, European Association of EchocardiographyRecommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology.J Am Soc Echocardiogr. 2005; 18: 1440-1463Abstract Full Text Full Text PDF PubMed Scopus (9443) Google Scholar After 4 weeks of Ang II infusion, animals were euthanized by carbon dioxide and perfused with 0.9% saline, followed by 10% formalin. The hearts were carefully dissected immediately after the perfusion, weighed, and fixed in 10% formalin for 24 hours. Hearts were transversely divided into sections at midventricle along the short axis (ie, perpendicular to the long axis), and the heart was embedded in paraffin or OCT compound. Sections (5 μm thick) were stained with hematoxylin and eosin, Masson's trichrome for collagen, and Prussian blue for hemosiderin. Quantitative image analysis will be performed using Image-Pro Plus 6.3 (Media Cybernetics, Rockville, MD) software, as described previously.22Cheung C.T. Deisher T.A. Luo H. Yanagawa B. Bonigut S. Samra A. Zhao H. Walker E.K. McManus B.M. Neutralizing anti-4-1BBL treatment improves cardiac function in viral myocarditis.Lab Invest. 2007; 87: 651-661Crossref PubMed Scopus (14) Google Scholar Tissue sections were stained and quantified, as described previously.23Hiebert P.R. Wu D. Granville D.J. Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice.Cell Death Differ. 2013; 20: 1404-1414Crossref PubMed Scopus (38) Google Scholar Toluidine Blue at pH 2.0 was used to stain mast cells. Immunohistochemistry was performed for GzmB (ab4059; Abcam, Cambridge, MA), vimentin (number 5741; Cell Signaling, Beverly, MA), fibroblast-specific protein-1 (ab27957; Abcam), α-smooth muscle actin (ab5694; Abcam), and cleaved caspase 3 (number 9661; Cell Signaling). Immunofluorescence staining was performed for CD45 (number 550539; BD Biosciences, Franklin Lakes, NJ), CD68 (MCA1957; AbD Serotec, Raleigh, NC), CD3 (ab5690; Abcam), CD31 (number 553370; BD Biosciences), and vascular endothelial (VE)-cadherin [ab33168 (Abcam) and AF1002 (R&D Systems, Minneapolis, MN)]. TdT in Situ Apoptosis Detection Kit (4810-30-K; R&D Systems) was used, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining was performed according to the manufacturer's manual. Cardiomyocyte size was determined by measuring cell diameter in sections of the left ventricular myocardium stained with wheat germ agglutinin coupled to Alexa Fluor 633 (W21404; Invitrogen, Burlington, ON, Canada), as described previously.24Ucar A. Gupta S.K. Fiedler J. Erikci E. Kardasinski M. Batkai S. Dangwal S. Kumarswamy R. Bang C. Holzmann A. Remke J. Caprio M. Jentzsch C. Engelhardt S. Geisendorf S. Glas C. Hofmann T.G. Nessling M. Richter K. Schiffer M. Carrier L. Napp L.C. Bauersachs J. Chowdhury K. Thum T. The miRNA-212/132 family regulates both cardiac hypertrophy and cardiomyocyte autophagy.Nat Commun. 2012; 3: 1078Crossref PubMed Scopus (447) Google Scholar, 25Coelho-Filho O.R. Shah R.V. Mitchell R. Neilan T.G. Moreno Jr., H. Simonson B. Kwong R. Rosenzweig A. Das S. Jerosch-Herold M. Quantification of cardiomyocyte hypertrophy by cardiac magnetic resonance: implications for early cardiac remodeling.Circulation. 2013; 128: 1225-1233PubMed Google Scholar Confocal images were acquired with a Leica AOBS SP2 laser-scanning confocal microscope (Leica, Heidelberg, Germany) and Leica Confocal Software TCS SP2 version 2.61 build 1537. Images were analyzed using Volocity three-dimensional image analysis software version 5.2.1 build 0 (PerkinElmer, Waltham, MA). Total RNA was extracted from hearts using formalin-fixed, paraffin-embedded Total RNA Isolation Kit (Invitrogen). cDNA was obtained by reverse transcribing a uniform amount of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Burlington, ON, Canada). The transcript levels of the genes of interest were measured by real-time PCR using the SYBR Green PCR mix (Applied Biosystems) in an Applied Biosystems 7300 detection system (Biorad, Mississauga, ON, Canada). The quality of the quantitative PCR run was determined by standard curves and melting curve analysis. The data were normalized to the expression of a cellular housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase. Primers sequences (forward and reverse) used in this study are listed in Table 2.Table 2Primer List for Real-Time PCRGene name (protein name)Forward sequenceReverse sequenceGene IDGzmb (GzmB)5′-CCACTCTCGACCCTACATGG-3′5′-GGCCCCCAAAGTGACATTTATT-3′14939Col1a1 (collagen I)5′-GAGCGGAGAGTACTGGATCG-3′5′-TACTCGAACGGGAATCCATC-3′12842Col3a1 (collagen III)5′-TGGTCCTCAGGGTGTAAAGG-3′5′-GTCCAGCATCACCTTTTGGT-3′12825Prf1 (perforin)5′-GTACAACTTTAATAGCGACACAGTA-3′5′-AGTCAAGGTGGAGTGGAGGT-3′18646Vim (vimentin)5′-CGTCCACACGCACCTACAG-3′5′-GGGGGATGAGGAATAGAGGCT-3′22352S100a4 (FSP-1)5′-TCCACAAATACTCAGGCAAAGAG-3′5′-GCAGCTCCCTGGTCAGTAG-3′20198Acta2 (α-SMA)5′-GTCCCAGACATCAGGGAGTAA-3′5′-TCGGATACTTCAGCGTCAGGA-3′11475Il1b (IL-1β)5′-GCAACTGTTCCTGAACTCAACT-3′5′-ATCTTTTGGGGTCCGTCAACT-3′16176Il6 (IL-6)5′-TAGTCCTTCCTACCCCAATTTCC-3′5′-TTGGTCCTTAGCCACTCCTTC-3′16193Tnf (TNF-α)5′-CCCTCACACTCAGATCATCTTCT-3′5′-GCTACGACGTGGGCTACAG-3′21926Ctgf (CTGF)5′-GAAGGGCAAAAAGTGCATCC-3′5′-GACAGTTGTAATGGCAGGCA-3′14219Tgfb1 (TGF-β)5′-CTCCCGTGGCTTCTAGTGC-3′5′-GCCTTAGTTTGGACAGGATCTG-3′21803Fn1 (fibronectin)5′-GATGTCCGAACAGCTATTTACCA-3′5′-CCTTGCGACTTCAGCCACT-3′14268Dcn (decorin)5′-TTCCTACTCGGCTGTGAGTC-3′5′-AAGTTGAATGGCAGAACGC-3′13179Mmp2 (MMP-2)5′-CAAGTTCCCCGGCGATGTC-3′5′-TTCTGGTCAAGGTCACCTGTC-3′17390Mmp9 (MMP-9)5′-CTGGACAGCCAGACACTAAAG-3′5′-CTCGCGGCAAGTCTTCAGAG-3′17395Gapdh (GAPDH)5′-AGGTCGGTGTGAACGGATTTG-3′5′-TGTAGACCATGTAGTTGAGGTCA-3′14433CTGF, connective tissue growth factor; FSP-1, fibroblast-specific protein-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GzmB, granzyme B; MMP, matrix metalloproteinase; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α. Open table in a new tab CTGF, connective tissue growth factor; FSP-1, fibroblast-specific protein-1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GzmB, granzyme B; MMP, matrix metalloproteinase; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; TNF-α, tumor necrosis factor-α. Mouse VE-cadherin (5 μg; 50192-M08H-50; Sino Biological, Beijing, China) was treated with or without 100 nmol/L recombinant mouse GzmB (G9278; Sigma-Aldrich) and incubated overnight at 30°C in GzmB assay buffer (50 mmol/L HEPES, pH 7.5, and 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate). For inhibition assays, 300 nmol/L of the murine serine protease inhibitor Serpin A3N (SA3N; generous gift from Dr. Chris R. Bleackley, University of Alberta, Edmonton, AB, Canada) was preincubated with 100 nmol/L GzmB for 1 hour at 30°C before the introduction of VE-cadherin. Reactions were stopped with the addition of SDS sample buffer (0.5 mol/L Tris, pH 6.8, 40% glycerol, 12% SDS, 20% dithiothreitol, and 0.25% bromophenol blue) and boiled for 5 minutes. Anti-mouse VE-cadherin antibody (AF1002; R&D Systems) was used for Western blot analysis. Human umbilical venous endothelial cells (HUVECs; CC-2517; Lonza, Basel, Switzerland) were analyzed by confocal microscopy after GzmB treatment, as described previously.26Ebnet K. Suzuki A. Horikoshi Y. Hirose T. Meyer Zu Brickwedde M.K. Ohno S. Vestweber D. The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM).EMBO J. 2001; 20: 3738-3748Crossref PubMed Scopus (327) Google Scholar Briefly, HUVECs grown on chamber slides (number 154453; Thermo Scientific, Rochester, NY) were incubated with 100 nmol/L recombinant human GzmB (Beryllium, Boston, MA) at 37°C for 24 hours. For inhibition assays, 100 nmol/L recombinant human GzmB was preincubated with 300 nmol/L SA3N at 37°C for 45 minutes before being added to the cells. Cells were fixed with 100% ice-cold methanol for 1 minute at room temperature. Cells were then permeabilized with phosphate-buffered saline containing 0.2% (v/v) Triton X-100 (Sigma-Aldrich, Oakville, ON, Canada) for 10 minutes and blocked in phosphate-buffered saline containing 1% (w/v) bovine serum albumin (Sigma-Aldrich, Oakville, ON, Canada) for 30 minutes at room temperature. VE-cadherin antibody (ab33168; Abcam) incubations were performed at room temperature in the above blocking solution for 1 hour at room temperature. DAPI was used for counterstain. Confocal images were acquired with a Leica AOBS SP2 laser-scanning confocal microscope (Leica) and Leica Confocal Software TCS SP2 version 2.61 build 1537. Transendothelial electrical resistance assays were performed as previously described.27Szulcek R. Bogaard H.J. van Nieuw Amerongen G.P. Electric cell-substrate impedance sensing for the quantification of endothelial proliferation, barrier function, and motility.J Vis Exp. 2014; : 51300Google Scholar Briefly, eight chambered 10E+ slides were coated with 10 mmol/L sterile L-cysteine (Applied Biophysics, Troy, NY) for 10 minutes before wells were washed with sterile water. HUVECs (Lonza) were plated at 1 × 105 cells per well in endothelial growth basal medium media supplemented with EGM-2 Singlequot kit (Lonza) for 0.5 hours at room temperature. Slides were then transferred to the Electric Cell-Substrate Impedance Sensing (Applied Biophysics) apparatus, and impedance, resistance, and capacitance were read continuously at multiple frequencies. After 48 hours in vitro, once cells had become confluent, medium with serum and growth factors was removed and replaced with serum-free EBM medium and returned to the apparatus for continuous read. After stabilizing (2 hours), 100 nmol/L recombinant human GzmB (Beryllium) was added. For SA3N inhibition, 100 nmol/L recombinant human GzmB (Beryllium) was incubated with 300 nmol/L SA3N at 37°C for 45 minutes before being added to the cells. Continuous measurements were then made for the following 72 hours. The average impedance and capacitance during a 1-hour period of time at specific time points were used for further data analysis. Quantitative values are expressed as means ± SEM. Statistical analysis was performed using GraphPad Prism version 5.01 (GraphPad Software, San Diego, CA). One-way analysis of variance with post hoc test or t-test was used, where appropriate, for group comparison analyses, with P < 0.05 considered statistically significant. To investigate the involvement of GzmB in cardiac fibrosis, diseased human cardiac tissue samples with established fibrosis were examined. Few GzmB-positive cells were observed in control healthy heart tissue, whereas GzmB-positive cells were prominent and diffusely present in fibrotic heart tissue (Figure 1A). Quantification revealed a similar expression pattern in all of the examined fibrotic heart samples regardless of differences in etiology, wherein the number of GzmB-positive cells was increased approximately threefold in comparison to normal hearts (Figure 1B). Using a murine model of Ang II–induced cardiac fibrosis, WT mice exhibited extensive cardiac fibrosis accompanied by elevated levels of GzmB (Figure 1C). Quantitative analysis showed that the number of GzmB-positive cells and the expression of GzmB mRNA were both increased approximately threefold in WT heart tissue after 4 weeks of Ang II treatment (Figure 1, D and E). To further investigate the cellular origin of GzmB in the heart, cardiac tissue sections were first stained with Toluidine Blue for mast cells, followed by immunostaining for GzmB. Our results showed that most GzmB-positive cells in the heart were mast cells (Figure 1F), which were also increased in the diseased hearts (Figure 1G). The costaining of GzmB and CD3 showed that some of the CD3-positive cells were GzmB positive. We observed no colocalization of CD68 and GzmB staining (Supplemental Figure S1). The elevated level of serum Ang II is correlated with the development of hypertension and cardiac hypertrophy.28Diz D.I. Baer P.G. Nasjletti A. Angiotensin II-induced hypertension in the rat: effects on the plasma concentration, renal excretion, and tissue release of prostaglandins.J Clin Invest. 1983; 72: 466-477Crossref PubMed Google Scholar Ang II–infused WT mice exhibited retarded weight gain during the 4-week treatment period, whereas saline-infused WT mice (saline control) continued to gain weight as expected. A retarded weight gain was also observed in Ang II–infused GzmB-deficient (Gzmb−/−) mice, which was similar to that of WT mice (Figure 2A). Ang II–induced hypertension was observed in WT and Gzmb−/− mice, with no difference observed between genotypes (Figure 2B). The heart rates for WT and Gzmb−/− mice were similar, and were not affected by Ang II infusion (Figure 2C). The impact of Ang II infusion on cardiac function and anatomy between WT and Gzmb−/− mice was further investigated via echocardiography.21Lang R.M. Bierig M. Devereux R.B. Flachskampf F.A. Foster E. Pellikka P.A. Picard M.H. Roman M.J. Seward J. Shanewise J.S. Solomon S.D. Spencer K.T. Sutton M.S. Stewart W.J. Chamber Quantification Writing Group, American Society of Echocardiography's Guidelines and Standards Committee, European Association of EchocardiographyRecommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology.J Am Soc Echocardiogr. 2005; 18: 1440-1463Abstract Full Text Full Text PDF PubMed Scopus (9443) Google Scholar Fractional shortening and ejection fraction are the two common measurements of cardiac function.29de Simone G. Devereux R.B. Roman M.J. Ganau A. Saba P.S. Alderman M.H. Laragh J.H. Assessment of left ventricular function by the midwall fractional shortening/end-systolic stress relation in human hyper
Abstract Background KRAS (KRAS proto-oncogene, GTPase; OMIM: 190,070) encodes one of three small guanosine triphosphatase proteins belonging to the RAS family. This group of proteins is responsible for cell proliferation, differentiation and inhibition of apoptosis. Gain-of-function variants in KRAS are commonly found in human cancers. Non-malignant somatic KRAS variants underlie a subset of RAS-associated autoimmune leukoproliferative disorders (RALD). RALD is characterized by splenomegaly, persistent monocytosis, hypergammaglobulinemia and cytopenia, but can also include autoimmune features and lymphadenopathy. In this report, we describe a non-malignant somatic variant in KRAS with prominent clinical features of massive splenomegaly, thrombocytopenia and lymphopenia. Case presentation A now-11-year-old girl presented in early childhood with easy bruising and bleeding, but had an otherwise unremarkable medical history. After consulting for the first time at 5 years of age, she was discovered to have massive splenomegaly. Clinical follow-up revealed thrombocytopenia, lymphopenia and increased polyclonal immunoglobulins and C-reactive protein. The patient had an unremarkable bone marrow biopsy, flow cytometry showed no indication of expanded double negative T-cells, while malignancy and storage disorders were also excluded. When the patient was 8 years old, whole exome sequencing performed on DNA derived from whole blood revealed a heterozygous gain-of-function variant in KRAS (NM_004985.5:c.37G > T; (p.G13C)). The variant was absent from DNA derived from a buccal swab and was thus determined to be somatic. Conclusions This case of idiopathic splenomegaly in childhood due to a somatic variant in KRAS expands our understanding of the clinical spectrum of RAS-associated autoimmune leukoproliferative disorder and emphasizes the value of securing a molecular diagnosis in children with unusual early-onset presentations with a suspected monogenic origin.
Primary atopic disorders are a group of inborn errors of immunity that skew the immune system toward severe allergic disease. Defining the biology underlying these extreme monogenic phenotypes reveals shared mechanisms underlying common polygenic allergic disease and identifies potential drug targets. Germline gain-of-function (GOF) variants in JAK1 are a cause of severe atopy and eosinophilia. Modeling the JAK1GOF (p.A634D) variant in both zebrafish and human induced pluripotent stem cells (iPSCs) revealed enhanced myelopoiesis. RNA-Seq of JAK1GOF human whole blood, iPSCs, and transgenic zebrafish revealed a shared core set of dysregulated genes involved in IL-4, IL-13, and IFN signaling. Immunophenotypic and transcriptomic analysis of patients carrying a JAK1GOF variant revealed marked Th cell skewing. Moreover, long-term ruxolitinib treatment of 2 children carrying the JAK1GOF (p.A634D) variant remarkably improved their growth, eosinophilia, and clinical features of allergic inflammation. This work highlights the role of JAK1 signaling in atopic immune dysregulation and the clinical impact of JAK1/2 inhibition in treating eosinophilic and allergic disease.
Rare diseases (RDs), more than 80% of which have a genetic origin, collectively affect approximately 350 million people worldwide. Progress in next-generation sequencing technology has both greatly accelerated the pace of discovery of novel RDs and provided more accurate means for their diagnosis. RDs that are driven by altered epigenetic regulation with an underlying genetic basis are referred to as rare diseases of epigenetic origin (RDEOs). These diseases pose unique challenges in research, as they often show complex genetic and clinical heterogeneity arising from unknown gene-disease mechanisms. Furthermore, multiple other factors, including cell type and developmental time point, can confound attempts to deconvolute the pathophysiology of these disorders. These challenges are further exacerbated by factors that contribute to epigenetic variability and the difficulty of collecting sufficient participant numbers in human studies. However, new molecular and bioinformatics techniques will provide insight into how these disorders manifest over time. This review highlights recent studies addressing these challenges with innovative solutions. Further research will elucidate the mechanisms of action underlying unique RDEOs and facilitate the discovery of treatments and diagnostic biomarkers for screening, thereby improving health trajectories and clinical outcomes of affected patients.
Abstract Background/Objectives The health care infrastructure of India, designed to treat acute problems, can benefit from preventive medicine-based policies that address chronic and non-communicable issues of relevance to India’s growing elderly population. Unintentional fall related injuries are one such issue whose economic burden can be streamlined with proper preventative public health policies. It is imperative that fall-risk factors specific to the Indian population be identified and analyzed for use in geriatric falls-risk assessment. We aim to determine factors predictive of falls in the aging Indian population in this study using Wave 1 data from the World Health Organization Study on Global Ageing and Adult Health (WHO SAGE) in India. Methods Cross-sectional analysis of results from WHO SAGE Wave 1 was conducted. Multivariate analysis was used to determine risk factors of falls specific to the Indian population in adults over the age of 50. Prediction models were created and evaluated using these risk factors and their performances were evaluated. SAGE Wave 1 India was implemented in six states that together provided nationally representative samples. Multistage stratified sampling was used to select these states and systematic sampling was used to select households from villages and urban districts within these states. Data from all individuals over the age of 50 in selected households was compiled for analysis. Findings 34 fall risk factors specific to the Indian population were determined. The model that did not weigh the factors was determined as the best model for possible use in clinically assessing old-age adults at risk for falling. Furthermore, 6 risk factors in the Indian census were used to identify falls risks hotspots on a district-level map of India. Conclusion This analysis can be used in public health policy recommendations and can form a basis for assessing and addressing falls-risk issues in India.
