Insufficiently Defined Genetic Background Confounds Phenotypes in Transgenic Studies As Exemplified by Malaria Infection in Tlr9 Knockout Mice

The use of genetically modified mice, i.e. transgenic as well as gene knockout (KO) and knock-in mice, has become an established tool to study gene function in many animal models for human diseases . However, a gene functions in a particular genomic context. This implies the importance of a well-defined homogenous genetic background for the analysis and interpretation of phenotypes associated with genetic mutations. By studying a Plasmodium chabaudi chabaudi AS (PcAS) malaria infection in mice bearing a TLR9 null mutation, we found an increased susceptibility to infection, i.e. higher parasitemia levels and increased mortality. However, this was not triggered by the deficient TLR9 gene itself. Instead, this disease phenotype was dependent on the heterogeneous genetic background of the mice, which appeared insufficiently defined as determined by single nucleotide polymorphism (SNP) analysis. Hence, it is of critical importance to study gene KO phenotypes on a homogenous genetic background identical to that of their wild type (WT) control counterparts. In particular, to avoid problems related to an insufficiently defined genetic background, we advocate that for each study involving genetically modified mice, at least a detailed description of the origin and genetic background of both the WT control and the altered strain of mice is essential

Mice Deficient in Nucleoporin Nup210 Develop Peripheral T Cell Alterations

The nucleopore is an essential structure of the eukaryotic cell, regulating passage between the nucleus and cytoplasm. While individual functions of core nucleopore proteins have been identified, the role of other components, such as Nup210, are poorly defined. Here, through the use of an unbiased ENU mutagenesis screen for mutations effecting the peripheral T cell compartment, we identified a Nup210 mutation in a mouse strain with altered CD4/CD8 T cell ratios. Through the generation of Nup210 knockout mice we identified Nup210 as having a T cell-intrinsic function in the peripheral homeostasis of T cells. Remarkably, despite the deep evolutionary conservation of this key nucleopore complex member, no other major phenotypes developed, with viable and healthy knockout mice. These results identify Nup210 as an important nucleopore complex component for peripheral T cells, and raise further questions of why this nucleopore component shows deep evolutionary conservation despite seemingly redundant functions in most cell types

Palaeogenomics of Upper Palaeolithic to Neolithic European hunter-gatherers

Publisher Copyright: © 2023, The Author(s).Modern humans have populated Europe for more than 45,000 years1,2. Our knowledge of the genetic relatedness and structure of ancient hunter-gatherers is however limited, owing to the scarceness and poor molecular preservation of human remains from that period3. Here we analyse 356 ancient hunter-gatherer genomes, including new genomic data for 116 individuals from 14 countries in western and central Eurasia, spanning between 35,000 and 5,000 years ago. We identify a genetic ancestry profile in individuals associated with Upper Palaeolithic Gravettian assemblages from western Europe that is distinct from contemporaneous groups related to this archaeological culture in central and southern Europe4, but resembles that of preceding individuals associated with the Aurignacian culture. This ancestry profile survived during the Last Glacial Maximum (25,000 to 19,000 years ago) in human populations from southwestern Europe associated with the Solutrean culture, and with the following Magdalenian culture that re-expanded northeastward after the Last Glacial Maximum. Conversely, we reveal a genetic turnover in southern Europe suggesting a local replacement of human groups around the time of the Last Glacial Maximum, accompanied by a north-to-south dispersal of populations associated with the Epigravettian culture. From at least 14,000 years ago, an ancestry related to this culture spread from the south across the rest of Europe, largely replacing the Magdalenian-associated gene pool. After a period of limited admixture that spanned the beginning of the Mesolithic, we find genetic interactions between western and eastern European hunter-gatherers, who were also characterized by marked differences in phenotypically relevant variants.Peer reviewe

Palaeogenomics of Upper Palaeolithic to Neolithic European hunter-gatherers

: Modern humans have populated Europe for more than 45,000 years1,2. Our knowledge of the genetic relatedness and structure of ancient hunter-gatherers is however limited, owing to the scarceness and poor molecular preservation of human remains from that period3. Here we analyse 356 ancient hunter-gatherer genomes, including new genomic data for 116 individuals from 14 countries in western and central Eurasia, spanning between 35,000 and 5,000 years ago. We identify a genetic ancestry profile in individuals associated with Upper Palaeolithic Gravettian assemblages from western Europe that is distinct from contemporaneous groups related to this archaeological culture in central and southern Europe4, but resembles that of preceding individuals associated with the Aurignacian culture. This ancestry profile survived during the Last Glacial Maximum (25,000 to 19,000 years ago) in human populations from southwestern Europe associated with the Solutrean culture, and with the following Magdalenian culture that re-expanded northeastward after the Last Glacial Maximum. Conversely, we reveal a genetic turnover in southern Europe suggesting a local replacement of human groups around the time of the Last Glacial Maximum, accompanied by a north-to-south dispersal of populations associated with the Epigravettian culture. From at least 14,000 years ago, an ancestry related to this culture spread from the south across the rest of Europe, largely replacing the Magdalenian-associated gene pool. After a period of limited admixture that spanned the beginning of the Mesolithic, we find genetic interactions between western and eastern European hunter-gatherers, who were also characterized by marked differences in phenotypically relevant variants

Proteolysis of systemic autoantigens and suppression of systemic autoimmunity by gelatinase B/MMP-9

Bij de mens omvat de familie van de matrix metalloproteïnasen (MMPs) 24verschillende neutrale endopeptidasen. Deze proteasen komen tussen infysiologische processen zoals reproductie, ontwikkeling, immuniteit enweefselherstel. Ontregeling van MMP activiteit heeft echter pathogene effecten bij kanker, inflammatie, vasculaire ziekten, neurodegeneratieve aandoeningen en auto-immuunziekten. Gelatinase B/MMP-9 wordt vooral gesecreteerd door macrofagen en neutrofiele granulocyten tijdens inflammatie, maar wordt ook geproduceerd door veel andere immuun- en kankercellen. Proteolyse van auto-antigenen door MMP-9 genereert immunodominante restepitopen voor auto-reactieve T cellen. Dit werdreeds aangetoond voor de klieving van collageen type II in reumatoïde artritis,van myelin basic protein en αB-crystalline in multiple sclerose, en vaninsuline in diabetes type I. Bovendien bleken MMP-9-deficiënte muizen minder gevoelig voor het ontwikkelen van symptomen in diermodellen van deze orgaanspecifieke auto-immuunziekten. Bijgevolg werd de vraag gesteld of MMP-9 ook systemische auto-antigenen kan klieven en hoe dit de ontwikkeling van systemischeauto-immuniteit zou beïnvloeden.Orgaan-specifieke auto-immuniteit is meestal gericht tegen een beperktaantal auto-antigenen die voorkomen in het aangetaste orgaan. Systemischeauto-immuunziekten worden daarentegen gekenmerkt door een breed spectrum aanauto-antigenen, vooral bestaande uit veel voorkomende intracellulaire eiwitten.Systemische lupus erythematosus of SLE is het type-voorbeeld van eensystemische auto-immuunziekte en wordt gekenmerkt door hoge titers vanauto-antistoffen tegen meer dan 100 verschillende intracellulaire and intranucleaireauto-antigenen. De vorming en afzetting van immuuncomplexen veroorzaaktinflammatie en weefselschade over het hele lichaam en tast vooral huid, nieren,gewrichten, longen, centraal zenuwstelsel en bloedvaten aan. In SLE veroorzaken genetische en omgevingsfactoren (bv. virale infecties, medicatie, toxines) een toename vanapoptose of geprogrammeerde celdood. Bovendien zijn verschillende mechanismenvoor het opruimen van apoptotische cellen verstoord. Bijgevolg zal de overmaat aan apoptotische cellen leiden tot secundaire necrose en openbarsten. Hierdoorworden intracellulaire eiwitten vrijgesteld in het extracellulair milieu, waar ze kunnen gemoduleerd worden door MMPs en andere proteasen, hetgeen een extra argument vormt voor de studie van MMP-9 in systemische auto-immuniteit.Om te bepalen of MMP-9 in staat is om systemische auto-antigenen teklieven, werd een systematische karakterisatie van het intracellulairesubstraatrepertoire of degradoom van MMP-9 opgestart aan de hand van een1-dimensionele degradomics methode. Om het necrotisch vrijstellen van intracellulaire eiwitten in SLE na te bootsen, werd een modelsysteem gebruikt van necrotische humane monocytische THP-1 cellen,met of zonder toevoeging van geactiveerd MMP-9. Op deze manier werd adenylyl cyclase-associated protein-1 of CAP1 geïdentificeerd als een nieuw enuitzonderlijk efficiënt substraat van MMP-9. CAP1 is een cytoskeleteiwit en een auto-antigeen in reumatoïde artritis en SLE. Bovendien, detecteerden we intact CAP1 in urines van patiënten met systemische auto-imuunziekten zoals SLE, Sjögrens syndroom en vasculitis, en in urines van gezonde personen. Verder werden geactiveerde vormen van MMP-9 teruggevonden in urinestalen van patiënten met klinische parameters van nierfalen, terwijl in urines van personen zonder nierziekte geen actief MMP-9 aanwezig was. Bovendien, werd in sommige urines van patiënten een omgekeerde relatie teruggevonden tussen de niveaus van intact CAP1 en van geactiveerd MMP-9, hetgeen suggereert dat CAP1 in vivo wordt afgebroken.Identificatie van bijkomende substraten was door de complexiteit van heteiwitmengsel niet mogelijk met een 1-dimensionele aanpak. Om op grotere schaalintracellulaire MMP-9 substraten te kunnen identificeren, werd eenvoudige engoedkope multidimensionele degradomics technologie ontwikkeld. Dezemultidimensionele methoden reduceren de complexiteit van het proteïnemengseldoor de eiwitten eerst te scheiden volgens hun netto-lading of isoëlektrischpunt met behulp van ionenuitwisselingschromatografie en vervolgens in eentweede dimensie volgens moleculair gewicht met behulp van SDS-PAGE encentrifugale filtratie. Toepassing van de multidimensionele degradomics technologie op THP-1 cytosol leidde tot de isolatie en visualisatie van ongeveer 200 kandidaatsubstraten waarvan een 70-tal geïdentificeerd werden m.b.v. tandem massaspectrometrie (MS/MS). Ongeveer 2/3 van deze kandidaatsubstraten was reeds bekend als auto-antigeen in verschillende auto-immuunziekten en kankers. Ook werden talrijke intracellulaire matrix eiwitten, zoals actine en tubuline, geïdentificeerd als nieuwe substraten van MMP-9. Deze resultatentoonden aan dat proteolyse van systemische auto-antigenen wel degelijk eenfunctie van MMP-9 kan zijn. Bovendien leidden deze data tot de suggestie dat proteolyse door MMP-9 vereist zou kunnen zijn om de overmaat aan toxische, immunogene intracellulaire (matrix) eiwitten en systemische auto-antigenen op te ruimen die worden vrijgesteld na extensieve necrose.Om de rol van MMP-9 in systemische auto-immuniteit in vivo te onderzoeken, werden muizengegenereerd die genetisch deficiënt zijn in MMP-9 en met de inactiverende lpr (lymfoproliferatie) mutatie in de apoptose-inducerende receptor Fas. C57Bl/6 muizen zonder functionele Fas receptor (B6lpr/lpr muizen) ontwikkelen matige lymfoproliferatie enchronische systemische auto-immuniteit op latere leeftijd en met weinigimmunopathologie. De additionele genetische knockout van MMP-9 in B6lpr/lpr muizen resulteerde echter in een versnelde en sterk toegenomen lymfoproliferatie met uitgesprokenlymfadenopathie en splenomegalie, en significant gereduceerde overlevingvergeleken met enkel deficiëntie van Fas. Bovendien leidde het ontbreken van MMP-9 in B6lpr/lpr muizen tot een verhoogde productie van auto-antistoffen tegen multipele auto-antigenen en tot meer uitgesproken auto-immune weefselschade. Vermits intacte auto-antigenen betere stimuli bleken te zijn voor auto-antilichaamproductie, zou de onderdrukking van lpr-geïnduceerde systemische auto-immuniteit een gevolg kunnen zijn van de opruiming van immunodominante T- en B-cel epitopen in auto-antigenische substraten. Bijgevolg zou het ontbreken van MMP-9-gemedieerde proteolyse tot een bijkomend defect in opruiming kunnen leiden, hetgeen de ontwikkeling van systemische auto-immuniteit kan versnellen en bestaande auto-immuunreacties kan amplificeren.We kunnen dus besluiten dat MMP-9 een uitgebreid spectrum aan intracellulaire (matrix) proteïnen en systemische auto-antigenen kan klieven. Opruiming van intracellulaire eiwitten zou cruciaal kunnen zijn om immuuntolerantie te behouden na acute of chronische necrose, zoals voorkomendbij SLE. Bovendien werd MMP-9 geïdentificeerd als een beschermende factor ineen in vivo model van systemische auto-immuniteit. Deze pre-klinische studies hebben medische implicaties. Verschillende studies stellen immers voor om MMP remmers te gebruiken in SLE en andere systemische auto-immuunziekten. Onze data suggereren echter datmen voorzichtig moet omspringen met de studie van MMP inhibitie bij SLE, aangezien hierdoor systemische auto-immuunreacties kunnen ontstaan of geamplificeerd worden in patiënten met een ongunstige genetische achtergrond.status: publishe

Intracellular substrate cleavage: a novel dimension in the biochemistry, biology and pathology of matrix metalloproteinases

Matrix metalloproteinases (MMPs), originally discovered to function in the breakdown of extracellular matrix proteins, have gained the status of regulatory proteases in signaling events by liganding and processing hormones, cytokines, chemokines, adhesion molecules and other membrane receptors. However, MMPs also cleave intracellular substrates and have been demonstrated within cells in nuclear, mitochondrial, various vesicular and cytoplasmic compartments, including the cytoskeletal intracellular matrix. Unbiased high-throughput degradomics approaches have demonstrated that many intracellular proteins are cleaved by MMPs, including apoptotic regulators, signal transducers, molecular chaperones, cytoskeletal proteins, systemic autoantigens, enzymes in carbohydrate metabolism and protein biosynthesis, transcriptional and translational regulators, and proteins in charge of protein clearance such as lysosomal and ubiquitination enzymes. Besides proteolysis inside cells, intracellular proteins may also be modulated by MMPs in the extracellular milieu. Indeed, many intracellular proteins exit cells by non-classical secretion mechanisms or by various conditions of cell death by apoptosis, necrosis and NETosis, and become accessible to extracellular proteases. Intracellular substrate proteolysis by MMPs is involved in innate immune defense and apoptosis, and affects oncogenesis and pathology of cardiac, neurological, protein conformational and autoimmune diseases, including ischemia-reperfusion injury, cardiomyopathy, Parkinson's disease, cataract, multiple sclerosis and systemic lupus erythematosus. Since the same MMP may affect physiology and pathology in different and even opposite ways, depending on its extracellular or subcellular localization, an additional layer of complexity is added to therapeutic MMP inhibition. Hence, further elucidation of intracellular MMP localizations and intracellular substrate proteolysis is a new challenge in MMP research.status: publishe

The biochemical, biological, and pathological kaleidoscope of cell surface substrates processed by matrix metalloproteinases

Matrix metalloproteinases (MMPs) constitute a family of more than 20 endopeptidases. Identification of specific matrix and non-matrix components as MMP substrates showed that, aside from their initial role as extracellular matrix modifiers, MMPs play significant roles in highly complex processes such as the regulation of cell behavior, cell-cell communication, and tumor progression. Thanks to the comprehensive examination of the expanded MMP action radius, the initial view of proteases acting in the soluble phase has evolved into a kaleidoscope of proteolytic reactions connected to the cell surface. Important classes of cell surface molecules include adhesion molecules, mediators of apoptosis, receptors, chemokines, cytokines, growth factors, proteases, intercellular junction proteins, and structural molecules. Proteolysis of cell surface proteins by MMPs may have extremely diverse biological implications, ranging from maturation and activation, to inactivation or degradation of substrates. In this way, modification of membrane-associated proteins by MMPs is crucial for communication between cells and the extracellular milieu, and determines cell fate and the integrity of tissues. Hence, insights into the processing of cell surface proteins by MMPs and the concomitant effects on physiological processes as well as on disease onset and evolution, leads the way to innovative therapeutic approaches for cancer, as well as degenerative and inflammatory diseases.status: publishe

Multidimensional degradomics identifies systemic autoantigens and intracellular matrix proteins as novel gelatinase B/MMP-9 substrates

The action radius of matrix metalloproteinases or MMPs is not restricted to massive extracellular matrix (ECM) degradation, it extends to the proteolysis of numerous secreted and membrane-bound proteins. Although many instances exist in which cells disintegrate, often in conjunction with induction of MMPs, the intracellular MMP substrate repertoire or degradome remains relatively unexplored. We started an unbiased exploration of the proteolytic modification of intracellular proteins by MMPs, using gelatinase B/MMP-9 as a model enzyme. To this end, multidimensional degradomics technology was developed by the integration of broadly available biotechniques. In this way, 100-200 MMP-9 candidate substrates were isolated, of which 69 were identified. Integration of these results with the known biological functions of the substrates revealed many novel MMP-9 Substrates from the intracellular matrix (ICM), such as actin, tubulin, gelsolin, moesin, ezrin, Arp2/3 complex subunits, filamin B and stathmin. About 2/3 of the identified candidates were autoantigens described in multiple autoimmune conditions and in cancer (e.g. annexin 1, nucleolin, citrate synthase, HMGB1, alpha-enolase, histidyl-tRNA synthetase, HSP27, HSC70, HSP90, snRNP D3). These findings led to the insight that MMPs and other proteases may have novel (immuno)regulatory properties by the clearance of toxic and immunogenic burdens of abundant ICM proteins released after extensive necrosis. In line with the extracellular processing of organ-specific autoantigens, proteolysis might also assist in the generation of immunodominant 'neo-epitopes' from systemic autoantigens. The study of proteolysis of ICM molecules, autoantigens, alarmins and other crucial intracellular molecules may result in the discovery of novel roles for proteolytic modification.status: publishe

Lpr-induced systemic autoimmunity is unaffected by mast cell-deficiency

The function of mast cells in allergic and organ-specific autoimmune responses is highly controversial. In the current study we aimed to dissect the role of mast cells in systemic autoimmunity in the B6(lpr/lpr) mouse, a spontaneous model of systemic lupus erythematosus. B6(lpr/lpr) mice were interbred with C57Bl/6-Kit(W-sh/W-sh) (Wsh) mice, resulting in mast cell-deficiency. The offspring from this cross (Lpr/Wsh mice) developed symptoms of lupus of the same severity as B6(lpr/lpr) mice. Loss of mast cells on the Lpr background did not alter autoantibody production, proteinuria, the composition of T and B cell populations or autoimmune pathology. Reduced c-Kit expression did drive expanded splenomegaly and impeded IL-4 production by by CD4(+) cells, suggesting minor functions for mast cells. In general we conclude that mast cell deficiency and c-Kit deficiency do not play a role in the pathogenesis of lupus in B6(lpr/lpr) mice.Immunology and Cell Biology accepted article preview online, 07 April 2015. doi:10.1038/icb.2015.49.status: publishe

Treatment patterns in moderate-to-severe plaque psoriasis : results from a Belgian cross-sectional study (DISCOVER)

Purpose: The present study aimed to evaluate current treatment patterns and achievement of treatment goals in Belgian patients with moderate-to-severe plaque psoriasis. Materials and methods: This cross-sectional observational study (DISCOVER) was conducted in 2011 - 2012 in Belgian dermatology centers. Patient data were collected during a single visit and included information on psoriasis management and severity (PASI and DLQI). Treatment success was defined according to the current European consensus treatment goal algorithm. Results: Of the 556 patients included in the study, 38.1% reported no current treatment or only topicals, 34.2% were being treated with traditional systemics and/or phototherapy, and 29.5% with biologics. Methotrexate (11.7%) was the most commonly prescribed traditional systemic and adalimumab (14.2%) was the most commonly prescribed biologic agent at the time of the study. The percentage of patients achieving treatment goals was significantly higher in biologic-treated patients (73.1%) compared to those using traditional systemics (50.6%), phototherapy (41.1%), or no treatment/only topicals (20.9%; p<.001). Conclusions: Nearly 40% of Belgian patients with moderate-to-severe psoriasis in the DISCOVER study were undertreated despite the severity of their disease. Undertreatment of psoriasis remains a problem in Belgium and more effective educational strategies are needed to ensure the best treatment outcome for these patients