Abstract Rheumatoid arthritis (RA) is a systemic autoimmune disease with symmetrical articular manifestations. The etiology of the disease is unknown. The prevalence of RA is approximately 0.5–1.0% in adults. In Finland, the annual incidence is 39/100 000. RA is about three times more common in females than in males. Most commonly the disease affects first the joints of feet and fingers. Chronic inflammation leads to erosions of cartilage, bone and tendons and may destroy the whole joint. The diagnosis of RA is mainly based on the clinical features of the disease. The American College of Rheumatology (ACR) 1987 revised classification criteria of RA have commonly been used for diagnosis. No specific diagnostic test is available. Rheumatoid factor (RF) has traditionally been used in the diagnosis, but only 70 to 80% of RA patients have RF in their serum. Other antibodies found in RA are the antiperinuclear factor (APF), the anti-keratin antibody (AKA) and the antibodies to cyclic citrullinated peptide (CCP), which recognize the citrulline-containing antigenic filaggrin protein. Citrulline is an amino acid that is post-translationally formed from arginine by peptidylarginine deiminase enzymes (PADs). Autoantibodies to citrullinated proteins are more specific for RA than RF. There is no filaggrin in joints, which indicates that the autoantibodies reacting with this protein most probably only reflect immunological cross-reaction. It has been postulated that autoimmunity against collagens might be involved in the pathogenesis of RA. There are antibodies binding to collagen in cartilage (type II collagen) and in bone (type I collagen). They have been tested by using collagen preparations rendered soluble by pepsin digestion. This digestion removes the carboxyterminal (C-terminal) telopeptides of collagen. Autoantibodies to the C-telopeptides of type I and II collagens were studied in this doctoral research. Autoantibodies to the citrullinated C-telopeptides of type I and II collagens were found in the serum of patients with RA. ELISA, CLIA and inhibition ELISA were used to detect these autoantibodies. Automatic CLIA gives a more than twofold number of positive findings compared to previous ELISA. Currently the best method for the detection of these autoantibodies is inhibition ELISA. These autoantibodies are specific for citrulline in the peptide sequence. Autoantibodies that bind the normal C-telopeptides of type I and II collagens were not inhibited by soluble normal or citrullinated telopeptides. However, the antibodies that bind only citrullinated telopeptides could be inhibited by corresponding citrullinated telopeptides. Autoantibodies binding the citrullinated telopeptides of type II collagen and anti-CCP predict synergistically the development of seropositive RA. / Tiivistelmä Nivelreuma (arthritis rheumatoides) on krooninen autoimmuunisairaus, jonka aiheuttajaa ei tunneta. Nivelreuman esiintyvyys aikuisilla on 0.5–1.0 prosenttia. Siihen sairastuu vuosittain 39/100 000 suomalaista aikuista. Naiset sairastavat nivelreumaa kolme kertaa yleisemmin kuin miehet. Sairaus alkaa tavallisesti päkiöistä ja sormien nivelistä. Nivelreuma aiheuttaa ruston, luun ja nivelsiteiden syöpymistä ja voi lopulta tuhota koko nivelen. Nivelreuman diagnoosi perustuu pääasiassa taudin kliinisiin piirteisiin. Yhdysvaltain reumajärjestön (American College of Rheumatology, ACR) vuonna 1987 esittämät luokittelukriteerit ovat yleisesti käytössä. Taudin toteamiseen ei ole spesifistä laboratoriotestiä. Perinteisesti käytettyä reumatekijää esiintyy 70–80 prosentilla potilaista. Muita nivelreumapotilaan seerumista esiintyviä vasta-aineita ovat antiperinukleriaaritekijä (APF), keratiinivasta-aineet (AKA) ja vasta-aineet sykliseen sitrullinisoituneeseen peptidiin (CCP). Sitrulliini on aminohappo, jonka peptidyyliarginiini deiminaasi -entsyymit (PAD) ovat muokanneet arginiinista posttranslationaalisesti. Sitrullinisoituneiden proteiinien autovasta-aineet ovat spesifisempiä nivelreumassa kuin reumatekijä. Nivelissä ei ole filaggriinia, mikä viittaa siihen, että todetut vasta-aineet perustuvat immunologiseen ristireaktioon. On epäilty, että nivelreuman patogeneesiin voisi liittyä autoimmuniteettia kollageeniin. Aiemmin tutkitut luun ja ruston (tyypin I ja II) kollageenivasta-aineet eivät ole olleet sitrullinisoituneita. Autovasta-ainetesteissä on käytetty pepsiinidigestiota, jolla kollageeni on saatu liukoiseksi. Pepsiinidigestio tuhoaa kuitenkin kollageenin karboksyyliterminaaliset (C-terminal) telopeptidit. Tässä väitöskirjatutkimuksessa tutkittiin tyypin I ja II kollageenien C-telopeptidejä. Nivelreumapotilaiden seerumissa todettiin autovasta-aineita, jotka sitoutuvat sitrullinisoituneisiin tyypin I ja II kollageeneihin. Todettuja vasta-aineita voidaan osoittaa ELISA-, CLIA- ja inhibitio-ELISA-menetelmillä. Automaattisella CLIA:lla saadaan kaksi kertaa enemmän positiivisia löydöksiä kuin aiemmin kehitetyllä ELISA:lla. Tällä hetkellä paras menetelmä näiden autovasta-aineiden osoittamiseen on inhibitio-ELISA. Todetut vasta-aineet ovat spesifisiä peptidin sekvenssissä olevaan sitrulliiniin. Vasta-aineita, jotka sitoutuvat normaaliin tyypin I ja II kollageenien C-telopeptidiin, ei voida inhiboida liukoisella normaalilla eikä sitrullinoidulla telopeptidillä. Kuitenkin vasta-aineita, jotka sitoutuvat vain sitrullinisoituihin telopeptideihin, voidaan inhiboida vastaavalla liukoisella sitrullinisoidulla telopeptidillä. Autovasta-aineet, jotka sitoutuvat samanaikaisesti sitrullinisoituneeseen tyypin II kollageenin C-telopeptidiin ja anti-CCP:hen ennustavat seropositiivista nivelreumaa.

There is a strong correlation between aging and risk of fractures that is independent of changes in bone quantity. Although the underlying processes that dysregulate the structural health and integrity of bone tissue without affecting bone-quantity with aging are unknown, one hypothesis is that changes affecting extra-cellular components of bone, particularly components of the mineralized matrix, may contribute to the decline in bone quality with aging. Citrullination is a post-translational modification where a peptidyl arginine is enzymatically modified to a peptidyl Citrulline by enzymes knowns as peptidyl arginine deiminases (PADs). Increased citrullination of proteins is associated with altered protein function and a loss of structural stability. We propose that citrullination could have negative effects on the structural integrity of bone matrix proteins by inducing a loss of proteostasis, a conditional loss of protein homeostasis. To address this, we examined whether the levels of citrullinated bone matrix proteins differs between young and old mice. Our results show that there is a profound increase in citrullination of extractable bone matrix with ageing. To assess if changes in citrullination contribute to bone mass and/or mechanical strength, we utilized an existing murine model in which citrullination is increased. Mice deficient in Protein Tyrosine Phosphatase Nonreceptor 22 (PTPN22), an inhibitor of PAD4, and thus of citrullination, have been shown to have increased histone H3 citrullination in macrophages derived from Ptpn22-/- mice compared to WT controls. We assessed several properties of bone in both young and aged Ptpn22-/- animals. In young mice, deficiency in PTPN22 leads to an increase in bone matrix citrullination. However, we did not find a link between increased citrullination and bone loss, as measured by formation of bone resorbing osteoclasts. Further, no difference in bone mass or structural parameters was seen in either young or old Ptpn22-/- mice compared to wild-type littermates. Interestingly, there was also no clear relationship between degree of citrullinated bone matrix and genotype in older mice, possibly due to overall increases in bone matrix citrullination with aging. However, as it is clear that bone matrix citrullination increases with age and that this citrullination in younger mice can be modulated by PTPN22 deficiency, we plan to use Ptpn22-/- mice as a model to learn more about the effects of increased citrullination on mechanical strength and bone matrix properties, therefore unearthing new insights into the role of post-translational modification in the function of bone matrix proteins.

Le complexe de remodelage de la chromatine NuRD, composé des sous-unités catalytiques CHD3 et CHD4, est un régulateur épigénétique de l’expression génique. Nos résultats montrent que NuRD s’associe avec les facteurs de transcription essentiels du mélanome que sont MITF et SOX10. Cependant, malgré une association physique et une co-localisation génomique, CHD4/NuRD ne semble pas agir comme un cofacteur important pour MITF ou SOX10. Néanmoins, la répression de CHD4 conduit à un ralentissement de la prolifération et déréprime l’expression des enzymes PADI1 et PADI3 dans les cellules de mélanome ainsi que dans de nombreux types de cellules cancéreuses. Ainsi, l’induction de ces enzymes, responsables de la conversion des arginines en citrullines, entraîne la citrullination spécifique de PKM2, une enzyme glycolytique essentielle, diminuant ainsi sa sensibilité aux inhibiteurs allostériques, et donc altérant l’équilibre physiologique entre activateurs et inhibiteurs de l’enzyme. L’ensemble de ce travail de thèse a permis de mettre en évidence une nouvelle voie reliant, d’une part la régulation épigénétique de l’expression de PADI1 et PADI3 par CHD4/NuRD ainsi que la reprogrammation de la régulation allostérique de PKM2 via la citrullination d’arginines, au flux glycolytique et au contrôle de la prolifération des cellules cancéreuses d’autre part. / The Nucleosome Remodelling and Deacetylation (NuRD) complex is an epigenetic regulator of gene expression that includes two mutually exclusive ATPase subunits CHD3 and CHD4. Our results show that NuRD associates with essential melanoma cell transcription factors namely MITF and SOX10. However, despite their physical association and genomic co-localization, CHD4-NuRD does not appear to act as a cofactor for MITF or SOX10 regulated gene expression. Nevertheless, CHD4 silencing leads to a slow growth phenotype and de-represses the expression of PADI1 (Protein Arginine DeIminase 1) and PADI3, two enzymes involved in converting arginines to citrullines in melanoma and multiple types of cancer cells. Increased expression of PADI1 and PADI3 enhances citrullination of arginines within the key glycolytic regulatory enzyme PKM2 then promoting excessive glycolysis, lowering ATP levels and slowing down proliferation. PKM2 citrullination lowers its sensitivity to allosteric inhibitors thus shifting equilibrium towards allosteric activators thereby bypassing the normal physiological regulation of glycolysis. Overall, our results lead to describe a novel pathway linking, epigenetic regulation of PADI1 and PADI3 expression by CHD4/NuRD and reprogramming of PKM2 allosteric regulation through arginines citrullination, to glycolytic flux and cancer cell proliferation.

Multiple sclerosis (MS) is a complex disease of the human central nervous system (CNS) involving the patchy destruction of the myelin sheath. Previous studies have found a consistent biochemical change in MS normal appearing white matter (NAWM) i.e. the increased citrullination of myelin basic protein (MBP) resulting in decreased myelin compaction. This process is facilitated by the enzyme family of peptidylarginine deiminases (PADs), of which PAD2 and PAD4 are expressed in mouse and human brain white matter. Therefore, we propose the inhibition of PAD enzymes would reverse protein hypercitrullination and represents a potential treatment for MS. Treatment with 2-chloroacetamidine (2CA), an active site inhibitor of PAD, attenuated diseases in four independent mouse models of MS associated with decreased PAD activity level, normalized peptidylcitrullination, and improved myelin morphology. Therefore, protein hypercitrullination may be a basic mechanism implicated in both neurodegenerative and autoimmune models of MS.

Multiple sclerosis (MS) is a complex disease of the human central nervous system (CNS) involving the patchy destruction of the myelin sheath. Previous studies have found a consistent biochemical change in MS normal appearing white matter (NAWM) i.e. the increased citrullination of myelin basic protein (MBP) resulting in decreased myelin compaction. This process is facilitated by the enzyme family of peptidylarginine deiminases (PADs), of which PAD2 and PAD4 are expressed in mouse and human brain white matter. Therefore, we propose the inhibition of PAD enzymes would reverse protein hypercitrullination and represents a potential treatment for MS. Treatment with 2-chloroacetamidine (2CA), an active site inhibitor of PAD, attenuated diseases in four independent mouse models of MS associated with decreased PAD activity level, normalized peptidylcitrullination, and improved myelin morphology. Therefore, protein hypercitrullination may be a basic mechanism implicated in both neurodegenerative and autoimmune models of MS.

Neutrophil extracellular traps (NETs), chromatin released by activated neutrophils, were first described for their antimicrobial properties. NETs have a backbone of DNA and histones lined with microbicidal proteins such as neutrophil elastase. NET release has pathological consequences, particularly within blood vessels where NETs can trap red blood cells and platelets, thus contributing to thrombosis (Chapter 1-Overview). NET formation (NETosis) is an active and coordinated biological process involving many enzymatic components. One enzyme in particular, peptidylarginine deiminase 4 (PAD4), citrullinates histones and is required for chromatin decondensation during NETosis. Neutrophils from PAD4-deficient mice are unable to form NETs. We obtained these mice from our collaborator Dr. Yanming Wang, and thus were able to compare PAD4-/- mice to wild-type (WT) mice in mouse models where NETs are formed. These studies have allowed for investigation of the biological relevance of PAD4 and NETs in vivo in thrombotic and/or inflammatory disease. This dissertation focuses on mouse models of deep vein thrombosis and of sepsis. In venous stenosis, thrombosis is initiated by restricting blood flow in the inferior vena cava (IVC). Here, PAD4-/- mice were greatly protected from thrombus formation (Chapter 2). Leukocyte rolling and platelet plug formation in response to vessel injury were unaffected, indicating that endothelial and platelet activation occurred normally in these mice. The mice did not exhibit any defects in hemostasis, and could be induced to produce deep vein thrombi by infusion of WT neutrophils that formed NETs as a part of the thrombus scaffold. Because there is potential to develop anti-NET therapies in thrombosis, I investigated if NET-deficiency would render mice immunocompromised (Chapter 3). PAD4-/- mice had similar mortality in the cecal ligation puncture model, and they were protected from shock in an LPS sepsis model where NETs are released in the absence of live bacteria. Therapies aimed at NET prevention or destruction would likely be beneficial without compromising host immunity. Thus, in summary, studying PAD4-deficient mice has revealed the impact of NETs in thrombotic/inflammatory disease and identified PAD4 as an attractive therapeutic target.

付記する学位プログラム名: 霊長類学・ワイルドライフサイエンス・リーディング大学院 / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第24837号 / 医博第5005号 / 新制||医||1068(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤, 能永, 教授 生田, 宏一, 教授 上野, 英樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Der Transkriptionsfaktor CCAAT/enhancer-binding protein α (C/EBPα) kontrolliert Zellzyklusarrest und terminale Differenzierung von neutrophilen Granulozyten und Adipozyten. Mutationen von C/EBPα treten häufig im Zusammenhang mit akuter myeloischer Leukämie auf. Massenspektrometrische Untersuchungen zeigten, dass C/EBPα an mehreren konservierten Argininen citrunilliert ist, einschließlich R297 in der C-terminalen basischen Region von C/EBPα. Mutationen von C/EBPα R297 wurden bereits beschrieben, weshalb der Schwerpunkt dieser Arbeit auf die Analyse der Modifikation dieses Aminosäurerestes gelegt wurde. Die Ergebnisse zeigen, dass die Peptidyl-Arginin-Deaminase (PADI4) mit C/EBPα interagiert und an mehreren Aminosäureresten citrunilliert. Citrunillierung oder Mutation von R297 beeinflusst die Aktivität von C/EBPα, einschließlich DNA-Bindung und Interaktion mit Partnerproteinen. Mutationsanalysen legen nahe, dass die positive Ladung des Aminosäurerestes R297 für die Bindung an cis-regulatorische DNA-Elemente, Protein Interaktionen, Genaktivierung, Fettzelldifferenzierung und Zellzyklusarrest ausschlaggebend ist. Knock-down von PADI4 in der myeloischen Vorläufer-Zelllinie 32D oder in der leukämischen U937 Zelllinie induziert Granulozyten-Differenzierung, möglicherweise durch Blockierung der PADI4-vermittelten Citrunillierung und Inaktivierung von C/EBPα. Zusammengefasst ergibt sich aus den Daten, dass PADI4 die positiv-geladene Seitenkette von C/EBPα R297 in eine ungeladene, citrunillierte Form umwandelt, die die Assoziation mit DNA destabilisiert und die C/EBPα-E2F-Interaktion beeinflusst, was wiederum das Gleichgewicht zwischen Proliferation und Differenzierung bestimmt. / The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) coordinates cell cycle arrest and terminal differentiation of neutrophil granulocytes and adipocytes. Mutations in C/EBPα are frequently associated with acute myeloid leukemia. Mass spectrometric analysis revealed that citrullination occurred on multiple conserved C/EBPα arginine residues including R297 in the C/EBPα basic region. C/EBPα R297 was previously reported to be mutated in acute myeloid leukemia and we therefore focused on the modification this residue. Data presented here show that peptidylarginine deiminase 4 (PADI4) interacts with and citrullinates C/EBPα at several sites. Citrullination or mutation of R297 dramatically changed C/EBPα activities, including DNA binding and interaction with protein partners. Mutational analysis demonstrated that the positive charge of residue R297 was critical for binding to cis-regulatory sites on DNA, gene activation, adipocytic differentiation, and cell cycle arrest. Knock down of PADI4 in the myeloid precursor cell line 32D or U937 leukemia cells induced granulocyte differentiation, potentially through relieving PADI4 mediated citrullination and inactivation of C/EBPα. Taken together, the data suggest that PADI4 converts the positive C/EBPα R297 side chain to the non-charged citrulline side chain which destabilizes the association with DNA and affects C/EBPα - E2F interaction that determines the balance between proliferation and differentiation.

A successful immune response depends on prompt and sufficient recruitment of leukocytes from the circulation to infected or injured sites. Mobilization of leukocytes to hypoxic tissues is vital for angiogenesis, i.e. the formation of new blood vessels from preexisting vasculature, and thus crucial for tissue growth and regeneration. Deviations from normal leukocyte recruitment drive a variety of pathologies, including chronic inflammation, autoimmune diseases and cancer, for which therapeutic options are limited or unspecific. Understanding the mechanisms by which the body controls leukocyte recruitment is therefore critical for the development of novel therapeutic strategies. The present investigations focused on delineating the mechanisms behind leukocyte mobilization from the bloodstream to afflicted sites, by means of in vivo imaging techniques and in vitro assays. We demonstrate that, in response to inflammation, increased vascular permeability enhances transendothelial transport of tissue-released chemokines. Within the vasculature, chemokines form a chemotactic gradient sequestered on heparan sulfate, which directs crawling neutrophils and expedites their extravasation to the inflamed tissue. Consequently, gradient formation grants efficient bacterial clearance. Citrullination of chemokines by leukocyte-derived PAD enzymes in the inflamed tissue prevents chemokine transport into blood vessels, which dampens further neutrophil recruitment and thereby controls the amplitude of the inflammatory response. Moreover, the mechanisms of neutrophil recruitment in response to proangiogenic factors released during hypoxia are revealed to differ from those observed during classical inflammation. Particularly, VLA-4 integrin and VEGFR1 expressed on a defined subset of neutrophils, along with endothelial VEGFR2, are required for efficient neutrophil recruitment to hypoxia. Rather than stimulus-induced phenotypic changes on neutrophils, specific neutrophil subtypes with innate proinflammatory or proangiogenic functions (respectively, CD49d-VEGFR1lowCXCR4low and CD49d+VEGFR1highCXCR4high) coexist in the circulation of humans and mice. In summary, this dissertation provides relevant information on specific steps of neutrophil recruitment to inflamed or hypoxic tissues, which may represent future means to down-regulate aberrant immune responses during chronic inflammation and autoimmune diseases; to increase angiogenesis during ischemia; or to limit pathological angiogenesis, a characteristic of tumor growth and of several chronic inflammatory disorders.

The degree of chromatin compaction plays a fundamental role in controlling the accessibility of DNA to the transcription machinery as well as other DNA-dependent biological pathways. The mammalian HP1 (Heterochromatin protein 1) protein family consists of three members: HP1α, β and γ. Each paralogue regulates formation and maintenance of heterochromatin by binding to the repressive chromatin marks H3K9me2/3 with their chromodomains (CDs). Despite high sequence conservation, each HP1 paralogue possesses specific functions, which are likely to be cell type specific. The aim of my thesis was to find novel functions for HP1γ in mouse embryonic stem cells (mESCs) and breast cancer cells. Mass spectrometry analysis identified citrullination of residues R38 and R39 within the CD of HP1γ. I show that these residues are citrullinated by peptidyl arginine deiminase 4 (PADI4) in vitro and in vivo. Mutations in HP1γ (R38/9A), designed to mimic the loss of charge accompanied with citrullination, affect HP1γ’s binding to H3K9me3 peptides and reduce its residence time on chromatin in differentiated mESCs, indicating a role for citrullination in regulating HP1γ binding to chromatin during differentiation. Furthermore, I studied the phenotype of HP1γ depletion in two human breast cancer models and found that HP1γ is essential for cell proliferation and viability of cancer, but not of normal epithelial cells. I performed whole transcriptome analysis in breast cancer cells depleted of HP1γ and cross-referenced it with its genomic localisation, which identified increased expression of interferon/antiviral defense genes and activation of pro-apoptotic pathways. Whilst genes involved in these pathways were not directly bound by HP1γ, this analysis also identified HP1γ as a novel regulator of zinc finger (ZNF) genes. In summary, I identified novel post-translational modifications in HP1γ and characterised them in mESCs. I further demonstrated a role for HP1γ regulating breast cancer cell viability and identified HP1γ as a novel regulator of ZNF genes. My findings highlight HP1γ as a potential target for breast cancer therapy.