Inflammasomi-tulehdussignalointireitin rooli valtimon rasvakovettumataudissa

Atherosclerosis is the underlying cause of myocardial infarction and stroke, the leading causes of death worldwide. It is a complex multifactorial disease closely linked with obesity, type II diabetes, and metabolic syndrome and, together, these conditions comprise the global epidemic of metabolic disorders that are becoming more and more prevalent, affecting adults and children alike. Atherosclerosis affects the large arteries that gradully loose their normal structure and function via a degenerative process involving lipid accumulation and chronic inflammation in the arterial wall. The lipid accumulation is driven by high circulating levels of cholesterol-carrying low density lipoproteins that become trapped and modified in the arterial wall. This causes an inflammatory reaction characterized by abundant immune cell infiltrates, mainly monocyte-derived macrophages. The macrophages scavenge large amounts of lipids and become activated to secrete a host of proinflammatory mediators and matrix-degrading enzymes that drive the progression of the disease. These processes result in the focal development of fatty lesions or plaques along the arteries. Over time, more complex lesions develop as a result of inflammatory and fibrotic responses, matrix remodeling, calcification, cholesterol crystallization, neovessel formation, and microhemorrhages. Ultimately, the plaques may rupture, causing thrombosis and acute complications. Although inflammation is recognized as a major driving force in atherosclerotic lesion development, the mechanisms triggering and maintaining the arterial wall inflammation remain incompletely understood. The aim of this thesis was to study the role of a key innate immune signaling pathway, the inflammasome, in atherosclerosis. The inflammasomes are large cytoplasmic signaling complexes that trigger the proteolytic maturation and secretion of two proinflammatory and proatherogenic cytokines, interleukin(IL)-1beta and -18. The inflammasome pathway can be triggered by microbial components or by sterile endogenous danger signals that elicit the activation of cytoplasmic sensor molecules from the NLR (nucleotide-binding domain and leucine-rich repeat containing) or PYHIN (pyrin and HIN domain containing) families. Despite the established roles of IL-1beta and -18 in driving atherosclerotic lesion development, the triggers of inflammasome activation in atherosclerotic plaques remained unknown. Macrophages are the prototypical inflammasome pathway-expressing cells, and thus cultured human macrophages were utilized to identify and characterize atherosclerosis-associated triggers of the inflammasome pathway. Cholesterol crystals and acidic environment were both found to trigger a strong inflammatory response via the activation of NLRP3 inflammasome and secretion of IL-1beta and IL-18. Cholesterol crystals are a hallmark of atherosclerotic lesions, yet they have been considered an inert material that merely acts as a sink for excess free cholesterol in the arterial wall. These new data suggested, however, that cholesterol crystals act as a potent sterile danger signal that may directly link pathological lipid accumulation and inflammation in the lesions. Local extracellular acidosis arises in the growing plaque due to the hindered diffusion of oxygen and the highly active glycolytic metabolism of macrophages. Acidic environment not only triggered the NLRP3 inflammasome, but even a very mild acidification from the physiological pH of 7.4 to 7.0 was sufficient to greatly amplify the IL-1beta response to other NLRP3 activators, including cholesterol crystals. Having showed that the atherosclerotic lesions harbour potent activators of the inflammasome pathway, we further analyzed the expression of this pathway in atherosclerotic human coronary specimens obtained from 10 explanted hearts. For this purpose, we utilized a quantitative PCR array targeting 88 inflammasome pathway-related molecules. Significant upregulation of 12 target genes was found in advanced coronary plaques compared to early lesions from the same coronary trees, including many of the very core components of the inflammasome pathway. Moreover, p38delta mitogen-activated protein kinase (MAPK), a poorly characterized isoform of the stress- and cytokine-activated p38 MAPK family, was consistently upregulated in advanced coronary plaques. Immunohistochemical stainings of human coronary lesions showed strong expression of NLRP3 inflammasome components and p38delta MAPK in macrophages surrounding the cholesterol crystal-rich lipid core. Furthermore, the p38delta MAPK was activated in cultured human macrophages upon NLRP3 inflammasome activation by cholesterol crystals and extracellular ATP, and required for NLRP3-mediated IL-1beta secretion. Taken together, the data presented in this thesis propose novel inflammasome-mediated mechanisms that may trigger sterile inflammation in atherosclerotic lesions and thus drive lesion progression.Tässä väitöskirjatyössä tutkittiin tulehdusreaktioiden merkitystä valtimon rasvakovettumataudin eli ateroskleroosin kehittymisessä. Ateroskleroosissa valtimoverisuonien seinämään kertyy sekä veren lipoproteiini-hiukkasten kantamaa kolesterolia että immuunipuolustuksen syöjäsoluja eli makrofageja. Makrofagit yrittävät poistaa liiallista kolesterolia, mutta juuttuvat lopulta rasvarakkuloiden täyttäminä valtimon seinämään ja laukaisevat ateroskleroosin etenemistä edistävän kroonisen tulehdusreaktion. Sen seurauksena rasva-aineiden ja tulehdussolujen muodostamat kertymät kehittyvät vähitellen monimuotoisiksi valtimoa ahtauttaviksi plakeiksi, jotka voivat revetessään aiheuttaa aivo- tai sydäninfarktin. Inflammasomi on makrofageista löydetty tulehdussignalointireitti, jonka aktivaatio laukaisee voimakkaan tulehdusreaktion käynnistämällä tulehdusvälittäjäaine interleukiini(IL)-1beta:n erityksen. Ateroskleroosin hiirimalleissa geneettinen IL-1beta-puutos vähentää huomattavasti plakkien kasvua, ja IL-1beta:n määrä lisääntyy myös ihmisen valtimon seinämässä plakkien kehittyessä. Plakkien inflammasomi-aktivaatiota ja IL-1beta-eritystä laukaisevia tekijöitä ei kuitenkaan aiemmin tunnettu ja väitöskirjatutkimuksen tavoitteena oli tunnistaa tällaisia tekijöitä. Tutkimus osoitti, että plakeissa yleisesti esiintyvät kolesterolikiteet sekä plakin kehittymiseen liittyvä kudosnesteen paikallinen happamoituminen laukaisevat makrofageissa voimakkaan inflammasomi-välitteisen tulehdusvasteen IL-1beta-erityksen kautta. Elimistön immuunipuolustus kykenee siis tunnistamaan kyseiset taudinkehitykseen liittyvät muutokset vaarasignaaleiksi ja reagoimaan niihin käynnistämällä tulehdusreaktion. Kolesterolikiteitä on vuosikymmenien ajan pidetty ateroskleroosin kehityksen kannalta merkityksettöminä sivutuotteina. Tutkimuksen tulokset haastoivat tämän käsityksen osoittamalla, että kolesterolikiteet ovat aktiivinen tekijä ateroskleroosin kehittymisessä ja voivat selittää häiriintyneen rasva-aineenvaihdunnan ja valtimon seinämän tulehduksen välistä yhteyttä. Lisäksi tutkimuksessa havaittiin, että jo hyvin lievä solunulkoinen happamoituminen voimistaa merkittävästi kolesterolikiteiden aiheuttamaa tulehdusvastetta makrofageissa, kun nämä ärsykkeet annetaan soluille samanaikaisesti. Väitöskirjatyössä inflammasomi-reitin toiminnallisuutta tutkittiin myös sydämen sepelvaltimonäytteissä laajan geeni-ilmentymisanalyysin ja vasta-ainevärjäysten avulla. Tulokset osoittivat, että kaikki inflammasomi-reitin keskeisimmät komponentit ilmentyvät sepelvaltimon seinämän makrofageissa ja useat niistä lisääntyvät merkittävästi ateroskleroottisten plakkien kehittymisen myötä. Geeni-ilmentymisanalyysissa havaittiin myös erään tulehdusta säätelevän molekyylin, p38delta MAP-kinaasin, lisääntyminen sepelvaltimoissa ateroskleroottisten plakkien kehittymisen myötä. Kyseistä molekyyliä tutkittiin tarkemmin viljellyissä ihmisen makrofageissa, mikä johti uuden tulehdusta säätelevän reitin löytymiseen. Tulokset osoittivat, että p38delta MAP-kinaasin aktivoituminen on keskeinen säätelijä kolesterolikiteiden laukaisemassa inflammasomi-aktivaatiossa. Ateroskleroottisten sairauksien hoito perustuu tällä hetkellä pääsääntöisesti veren kolesterolipitoisuutta alentavaan lääkitykseen. Valtimon seinämän tulehdusmekanismien tarkka selvittäminen luo perustan uudentyyppisen, kroonista tulehdustilaa hillitsevän lääkityksen kehittämiseen tämän kansanterveydellisesti merkittävän taudin hoitoon

Comparison of Tracheal Wash and Bronchoalveolar Lavage Cytology in 154 Horses With and Without Respiratory Signs in a Referral Hospital Over 2009−2015

Most equine lower respiratory diseases present as increased airway neutrophilia, which can be detected in tracheal wash (TW) or bronchoalveolar lavage fluid (BALE) cytology samples. The aim was to compare the TW and BALF results in a population of client-owned horses with and without clinical respiratory disease signs. A secondary aim was to determine the sensitivity (Se) and specificity (Sp) of TW and BALF neutrophilia in detecting respiratory disease. The cutoff values for neutrophils were also evaluated. Retrospective data from 154 horses of various breeds that had been subject to both TW and bronchoalveolar lavage (BAL) sampling at rest during 2009-2015 were used. The horses were divided into three groups based on the presenting signs, physical examination, and endoscopy mucus score. Neutrophil counts of >20% in TW and >5% in BAL were considered abnormal. Cytology results between groups, correlations between 1W and BALF cell types, and tracheal mucus score were analyzed. Two graph receiving operating characteristic (ROC) curves of the neutrophil percentage values of TW and BALF were created to determine the optimal cutoff values and to calculate the diagnostic Se and Sp for diagnosing airway inflammation in horses with and without clinical respiratory signs. The Se and Sp of TW and BALF neutrophil percentages were further estimated using a two-test one-population Bayesian latent class model. The two tests showed substantial agreement, and only 17.5% of the horses were classified differently (healthy vs. diseased). The neutrophil percentage was found to correlate between TW and BALF. The Se and Sp of TW were generally higher than for BAL when estimated with area under the curve or Bayesian model. Cutoff values of 17.7% for TW and 7% for BALF were indicated by the ROCs. We conclude that TW is a more sensitive and specific method in our patient population. We suggest that the current neutrophil cutoff values of 20% for 1W and 5% for BALE would still be appropriate to use in clinical diagnosis of airway inflammation. However, further studies with other cell types and in other populations are warranted to determine the best sampling method for individual horses.Peer reviewe

Tuotekehityksessä ja tutkimuksessa tarvittavien proteiinien tuottaminen kasveissa perunan A-viruksen avulla

Viruksien käyttö tuotekehityksen ja tutkimuksen vaatimien proteiinien tuottamiseen, syötävien rokotteiden kehittämiseen ja geeniterapiaan edustavat kasvavia biotekniikan sovellusalueita. Perunan A-virus (PVA) kuuluu potyviruksiin, joiden proteiinit tuotetaan aluksi yhtenä suurena molekyylinä. Se pilkotaan yksittäisiksi proteiineiksi viruksen itsensä tuottamilla entsyymeillä. Siten virusgenomiin lisätty vieras geeni käännetään proteiiniksi virusproteiinien mukana. Lopputuloksena sekä viruksenproteiineja että vierasta proteiinia tuotetaan kasvisoluissa samansuuruinen määrä. PVA:n proteiini-kuoren koontimekanismi sallii perintöaineksen merkittävän lisäyksen ilman, että viruksen tartutuskykymerkittävästi heikkenee. Koska virus monistuu ja leviää koko kasviin, jo melko pieni määrä kasveja riittää huomattavan proteiinimäärän tuottamiseen esimerkiksi säännösten mukaisessa kasvihuoneessa.Tämän työn tarkoituksena oli hyödyntää PVA:n genomia yhden vieraan proteiinin tai useiden erilaisten proteiinien samanaikaiseen tuottamiseen kasveissa. Aluksi kokeiltiin vieraan geenin kloonaamista viruksen replikaasia ja kuoriproteiinia koodaavien genomialueiden väliin. Työhön valittiin kaksi ihmisestä peräisi olevaa geeniä. Toinen geeneistä tuotti sorsiiniproteiinia, joka toimii sydänlihaksen supistuksen säätelijänä. Toinen puolestaan tuotti S-COMT-entsyymiä (katekoli-O-metyylitransferaasi), jonka aktiivisuuden rajoittaminen auttaa Parkinsonin taudin hoidossa. Kasvissa tuotettua S-COMT:ia voitaisiin käyttää lääkekehityksessä estolääkkeiden testaukseen. Kahden viikon kuluttua tartutuksesta tupakan lehdissä oli entsymaattisesti aktiivista S-COMT:ia n. 1 % lehden liukoisista proteiineista. Sorsiini sen sijaan oli pysymätön kasvisoluissa, sillä sitä ei havaittu mitattavia määriä. Tulos osoitti, että kaikki ihmisen proteiinit eivät sellaisenaan sovellu kasvissa tuotettaviksi.Transposonimutaatioon perustuneella tutkimuksella oli paikannettu PVA:n P1-proteiinia koodaavalta alueelta kohta, johon voitaisiin siirtää vieras geeni. Asia varmistettiin siirtämällä tähän kohtaan meduusan geeni, joka tuottaa UV-valossa vihreänä fluoresoivaa proteiinia (GFP). GFP-geeniä kantava PVA levisi kasvissa ja lisääntyi n. 30-50 %:iin viruksen normaalista pitoisuudesta. Koko kasvi fluoresoi vihreänä UV-valossa. Sama voitiin havaita myös tuottamalla GFP jo edellä mainitusta, ihmisen proteiinien tuottamiseen käytetystä PVA-genomin kohdasta.Vieras geeni voidaan sijoittaa myös potyviruksen P1- ja HCpro-proteiineja koodaavien alueiden väliin. Tämä on mahdollista myös PVA:ssa, mikä osoitettiin tässä työssä. Siten samaan PVA-genomiin voitiin siirtää kolme geeniä, yksi kuhunkin kolmesta kloonauskohdasta. Lopputuloksena oli PVA-genomi, jossa GFP-geeni sijaitsi P1:n sisällä, merivuokon lusiferaasigeeni P1/HCpro-kohdassa ja bakteerin beta-glukuronidaasigeeni (GUS) replikaasi/kuoriproteiinikohdassa. Virusgenomin ja itse viruksen pituudet kasvoivat 38 %, mutta virus säilytti tartutuskykynsä. Se levisi kasveissa saavuttaen 10-15 % viruksen normaalista pitoisuudesta. Kaikki kolme vierasta proteiinia tuotettiin huomattavina pitoisuuksina ja aktiivisina kasvien lehdissä

Vitamin C boosts DNA demethylation in TET2 mutation carriers

Background Accurate regulation of DNA methylation is necessary for normal cells to differentiate, develop and function. TET2 catalyzes stepwise DNA demethylation in hematopoietic cells. Mutations in the TET2 gene predispose to hematological malignancies by causing DNA methylation overload and aberrant epigenomic landscape. Studies on mice and cell lines show that the function of TET2 is boosted by vitamin C. Thus, by strengthening the demethylation activity of TET2, vitamin C could play a role in the prevention of hematological malignancies in individuals with TET2 dysfunction. We recently identified a family with lymphoma predisposition where a heterozygous truncating germline mutation in TET2 segregated with nodular lymphocyte-predominant Hodgkin lymphoma. The mutation carriers displayed a hypermethylation pattern that was absent in the family members without the mutation.Methods In a clinical trial of 1 year, we investigated the effects of oral 1 g/day vitamin C supplementation on DNA methylation by analyzing genome-wide DNA methylation and gene expression patterns from the family members.Results We show that vitamin C reinforces the DNA demethylation cascade, reduces the proportion of hypermethylated loci and diminishes gene expression differences between TET2 mutation carriers and control individuals.Conclusions These results suggest that vitamin C supplementation increases DNA methylation turnover and provide a basis for further work to examine the potential benefits of vitamin C supplementation in individuals with germline and somatic TET2 mutations.Peer reviewe

Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding

Background and aims: Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. Methods: Mature human MCs were differentiated from human peripheral blood-derived CD34(+) progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. Results: Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. Conclusions: The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Lasten ja nuorten tapaturmakuolleisuus on Suomessa yleisempää kuin Euroopassa keskimäärin : Tapaturmien ehkäisyä tehostettava

English summaryPeer reviewe

No evidence of EMAST in whole genome sequencing data from 248 colorectal cancers

Microsatellite instability (MSI) is caused by defective DNA mismatch repair (MMR), and manifests as accumulation of small insertions and deletions (indels) in short tandem repeats of the genome. Another form of repeat instability, elevated microsatellite alterations at selected tetranucleotide repeats (EMAST), has been suggested to occur in 50% to 60% of colorectal cancer (CRC), of which approximately one quarter are accounted for by MSI. Unlike for MSI, the criteria for defining EMAST is not consensual. EMAST CRCs have been suggested to form a distinct subset of CRCs that has been linked to a higher tumor stage, chronic inflammation, and poor prognosis. EMAST CRCs not exhibiting MSI have been proposed to show instability of di- and trinucleotide repeats in addition to tetranucleotide repeats, but lack instability of mononucleotide repeats. However, previous studies on EMAST have been based on targeted analysis of small sets of marker repeats, often in relatively few samples. To gain insight into tetranucleotide instability on a genome-wide level, we utilized whole genome sequencing data from 227 microsatellite stable (MSS) CRCs, 18 MSI CRCs, 3 POLE-mutated CRCs, and their corresponding normal samples. As expected, we observed tetranucleotide instability in all MSI CRCs, accompanied by instability of mono-, di-, and trinucleotide repeats. Among MSS CRCs, some tumors displayed more microsatellite mutations than others as a continuum, and no distinct subset of tumors with the previously proposed molecular characters of EMAST could be observed. Our results suggest that tetranucleotide repeat mutations in non-MSI CRCs represent stochastic mutation events rather than define a distinct CRC subclass.Peer reviewe

Haploinsufficiency of A20 impairs protein–protein interactome and leads into caspase-8-dependent enhancement of NLRP3 inflammasome activation

Objectives TNFAIP3 encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression. TNFAIP3 polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and, recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease ‘haploinsufficiency of A20’ (HA20). Here we describe a family with HA20 caused by a novel TNFAIP3 loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway.Methods NF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein–protein interactions of wild-type and TNFAIP3 p.(Lys91*) mutant A20 were analysed using mass spectrometry. NF-κB -dependent transcription, cytokine secretion and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.Results The protein–protein interactome of p.(Lys91*) mutant A20 was severely impaired, including interactions with proteins regulating NF-κB activation, DNA repair responses and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signalling, which led to increased NF-κB -dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients’ immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1β and interleukin-18.Conclusions The current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.Peer reviewe

Novel TMEM173 Mutation and the Role of Disease Modifying Alleles

Upon binding to pathogen or self-derived cytosolic nucleic acids cyclic GMP-AMP synthase (cGAS) triggers the production of cGAMP that further activates transmembrane protein STING. Upon activation STING translocates from ER via Golgi to vesicles. Monogenic STING gain-of-function mutations cause early-onset type I interferonopathy, with disease presentation ranging from fatal vasculopathy to mild chilblain lupus. Molecular mechanisms underlying the variable phenotype-genotype correlation are presently unclear. Here, we report a novel gain-of-function G207E STING mutation causing a distinct phenotype with alopecia, photosensitivity, thyroid dysfunction, and features of STING-associated vasculopathy with onset in infancy (SAVI), such as livedo reticularis, skin vasculitis, nasal septum perforation, facial erythema, and bacterial infections. Polymorphism in TMEM173 and IFIH1 showed variable penetrance in the affected family, implying contribution to varying phenotype spectrum. The G207E mutation constitutively activates inflammation-related pathways in vitro, and causes aberrant interferon signature and inflammasome activation in patient PBMCs. Treatment with Janus kinase 1 and 2 (JAK1/2) inhibitor baricitinib was beneficiary for a vasculitic ulcer, induced hair regrowth and improved overall well-being in one patient. Protein-protein interactions propose impaired cellular trafficking of G207E mutant. These findings reveal the molecular landscape of STING and propose common polymorphisms in TMEM173 and IFIH1 as likely modifiers of the phenotype.Peer reviewe