Bronchiectasis is a chronic inflammatory lung disease associated with failure of the normal mucociliary escalator, chronic bacterial colonisation of the airways, neutrophil mediated inflammation and a resulting clinical syndrome of respiratory infections, lung damage and symptoms such as cough, sputum production and shortness of breath. These are few effective treatments and the cause of bronchiectasis is unknown in the majority of patients. It is hypothesised that unrecognised immune defects may predispose to bronchiectasis or affect the severity of lung disease. Ficolin-2 is a circulating innate immune protein able to activate the lectin pathway of complement through interaction with mannose binding lectin associated serine protease-2. Through its structural and functional similarity to complement component C1q and mannose binding lectin, it is hypothesised that ficolin-2 may be involved in opsonophagocytosis of pathogens. A number of single nucleotide polymorphisms in the ficolin-2 gene have been described causing considerable variation in human ficolin-2 serum concentrations in healthy individuals. In this thesis, the role of the key lectin pathway components ficolin-2 and mannose binding lectin, are investigated in patients with bronchiectasis. We demonstrate a significant association between single nucleotide polymorphisms in the FCN2 gene and disease severity in bronchiectasis. Specifically, patients with low expressing FCN2 haplotypes have a higher frequency of chronic colonisation, colonisation with P. aeruginosa, more frequent exacerbations and worse health related quality of life. An association between MBL deficient genotypes and disease severity is also demonstrated suggesting an important role for the lectin pathway of complement in modifying disease severity in bronchiectasis. In-vitro studies identify that ficolin-2 is the major lectin pathway component responsible for complement activation on P. aeruginosa and that ficolin-2 binds to a wide range of clinically relevant pathogens. Neutrophils isolated from the sputum of patients with bronchiectasis showed significant alterations in surface receptor expression and function compared to peripheral blood neutrophils, with a novel effect of neutrophil elastase cleavage of CD88 contributing to reduced phagocytosis by airway neutrophils. Despite loss of phagocytic receptors from sputum neutrophils, opsonisation by ficolin-2/MASP-2 complexes still enhanced phagocytosis of P. aeruginosa by sputum neutrophils, suggesting that ficolin-2 may be relevant in the clearance of P. aeruginosa in the airway. In summary, ficolin-2 was found to be an important modifier of disease severity in bronchiectasis.

Mannose-binding lectin (MBL) and ficolins are complement-activating proteins, and both play an important role in innate immunity by recognizing specific carbohydrate moieties on the surface of wide range of microorganisms. Previous studies have shown that porcine ficolin-α and MBL-A bind to surface polysaccharides of bacteria cultured in suspension, but their interactions with bacteria in biofilm culture have not been studied. The objectives of this thesis were to determine whether porcine plasma ficolin and MBL bind to Actinobacillus pleuropneumoniae in biofilm cultures. APP serotype 5a (APP5a) was used because it produced pronounced biofilm in plastic culture dishes, in comparison with APP5b that was previously reported to bind ficolin in suspension cultures. N-acetylglucosamine (GlcNAc) in the biofilm produced by APP5a was stained with wheat germ agglutinin conjugated with Alexa Fluor-555 and identified by confocal laser scanning microscopy (CLSM). Dispersin B prevented APP5a biofilm formation indicating the requirement of poly N-acetylglucosamine (PNAG) for bacterial cohesion. Bound purified ficolin or ficolin in plasma both were eluted with GlcNAc from APP5a biofilm cultures. To address preferential binding of ficolin-α to biofilm matrix, ficolin-α was eluted with GlcNAc from extracellular polymeric substances (EPS) in supernatant after pelleting the bacteria. Biotinylated-ficolin that retained GlcNAc-binding activity for APP5b planktonic cultures was shown to bind strongly to APP5a biofilm, as detected by fluorescent NeutrAvidin staining and CLSM, but not in the presence of GlcNAc. Further, MBL-A in ficolin-depleted porcine plasma also bound to APP5a biofilm and was eluted with a sugar solution containing GlcNAc, galactose, mannose and glucose. These studies demonstrate that both porcine ficolin-α and MBL-A bind to biofilm cultures of APP5a in a carbohydrate-dependent manner, and suggest that the production of PNAG in biofilm is a binding target for ficolin. / Natural Sciences and Engineering Research Council of Canada (NSERC)

The complement system is a part of the innate immunity. Its function is to eliminate pathogens, by proteins interacting directly with pathogen surfaces and promoting a pro-inflammatory and anti-microbial environment. Related to the lectin pathway of the complement system are ten known proteins, with component properties and disease association still unclear. The aim of this study was to evaluate the instrument WES for the detection of seven proteins associated to the lectin pathway. The novel instrument introduces an automated technique based on capillary electrophoresis and immunoblotting. Trials were performed on donor plasma using instrument associated kits. For the evaluation, these kits were combined with assorted primary and secondary antibodies from several species, as well as antibodies in biotinylated form. The high protein content of plasma caused many artefacts, affecting separation and displaying unspecific binding of both primary and secondary antibodies. Biotinylated antibodies coupled with the kit streptavidin-horseradish peroxidase showed the best results for further trials. Several issues remain to be solved in the optimization, including determining the unspecificity of biotinylated primary antibodies, best antibody concentrations and optimal sample preparation and dilution.

Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus. In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro. Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity. In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP. Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish. / Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 737

Les collagènes de défense (C1q, MBL, ficolines) sont capables de reconnaître de nombreux motifs à la surface des éléments du non soi ou du soi altéré, via leurs domaines globulaires C-terminaux. Ils peuvent également interagir avec certains récepteurs présents à la surface des cellules humaines ou de pathogènes. Nous nous sommes intéressés dans un premier temps à la calréticuline de Trypanosoma Cruzi (TcCRT), une protéine qui interviendrait dans les mécanismes d'évasion de ce parasite. Dans le but de réaliser des études fonctionnelles et structurales de la TcCRT, nous avons produit différents fragments recombinants. Nous ne sommes cependant pas parvenus à obtenir un échantillon nous permettant d'accomplir nos objectifs, nous conduisant à reporter nos efforts sur l'étude d'un autre récepteur, CR1/CD35. Il a été montré précédemment que CR1/CD35 pouvait interagir avec C1q et la MBL, probablement par ses modules CCP 22-30. Cette interaction pourrait être impliquée dans l'élimination des complexes immuns, la phagocytose ou encore des mécanismes de signalisation cellulaire. A l'aide d'un fragment recombinant comprenant les modules CCP 22-30 de CR1, nous avons confirmé par SPR l'interaction avec C1q et la MBL. Nous avons également montré pour la première fois que CR1 pouvait interagir avec les ficolines L, H et M par ce même domaine. Nos résultats indiquent que cette interaction prendrait majoritairement place dans la région collagène de C1q, de la MBL et de la ficoline L, probablement à proximité du site de fixation des protéases. L'utilisation de fragments tronqués de CR1 CCP 22-30, nous permet de proposer l'hypothèse que les modules CCP 24 et 25 de CR1 seraient le site majoritaire de fixation des collagènes de défense. Ces données ouvrent la voie à des études structurales et fonctionnelles visant à approfondir notre connaissance des interactions CR1 – collagène de défense et de leur rôle physiologique. / The defence collagens (C1q, MBL, ficolins) are able to recognize various patterns on non-self or altered-self surfaces through their globular domains. They can also interact with receptors at the surface of human cells or pathogens. First, we were interested in the calreticulin from Trypanosoma cruzi, a protein which may be involved in the evasion mechanisms of that parasite. To achieve structural and functional studies, we produced recombinant fragments from TcCRT. Unfortunately, we couldn't obtain any sample suitable for our studies, so we decided to focus on another receptor, CR1/CD35. It has been shown previously by other teams that C1q and MBL bind to CR1/CD35, probably through CCP modules 22 to 30, close to the cell membrane. This interaction could be involved in several biologic mechanisms: elimination of immune complexes, phagocytosis, cell signaling. We produced a recombinant fragment including the CCP modules 22 to 30 of CR1 and confirmed its interaction with C1q and MBL using SPR. We also showed for the first time that L-, H- and M-ficolins bind to CR1 through CCP modules 22 to 30. Our results point out that the CR1 binding site of C1q, MBL and L-ficolin is located in the collagen stalks, most probably at or in close proximity to the serine protease interaction site. By using CR1 CCP 22-30 truncated fragments, we suggest that CCP modules 24 to 25 could be the main binding site for the defence collagens. These data open the way for structural and functional studies aiming at improving our knowledge of the CR1 – defense collagen interactions and of their physiological role.

Proteasomen, die die proteolytisch aktiven Untereinheiten LMP7, LMP2 und MECL1 inkorporieren, nennt man Immunoproteasomen. Während einer Immunreaktion führen diese regulierende sowie modulierende Funktionenaus. Sie sind konstitutiv exprimiert in Zellen hämatopoetischen Ursprungs, ein Bestandteil des angeborenen Immunsystems, die die erste Angriffsfront gegen pathogene Mikroorganismen ausbilden. Um die Bedeutung des Immunoproteasoms für die angeborene Immunantwort bei einer Streptococcus pneumoniae Infektion auf zu zeigen, charakterisierten wir den Krankheitsverlauf einer bakteriellen Pneumonie und analysierten lokale aber auch systemische Immunreaktionen in LMP7 ko Mäusen mit Hilfe eines S. pneumoniae Infektionsmodels. Die hier generierten Daten zeigten einen fortgeschrittenen Krankheitsverlauf in LMP7 ko Mäuse, der in einer systemischen inflammatorischen Immunreaktion endete und sich in klinischen Parametern, wie physiologische Kondition, spezifische diagnostische Marker und Immunsuppression, andeutete. Der Zustand der Sepsis entwickelte sich vermutlich aufgrund einer erhöhten bakteriellen Last im Blut und führte zu einer vorzeitigen Mortalität infizierter LMP7 ko Tiere. Obwohl die Fähigkeit von LMP7 ko Leukozyten ex vivo Bakterien zu eliminieren nicht beeinträchtigt war, zeigten LMP7 ko Mäuse in vivo eine verminderte Genexpression immunmodulierender Moleküle, wie Pentraxine, Fikoline und Kollektine. Diese Moleküle fördern die Aufnahme, Elimination und Degradation pathogener Mikroorganismen. Die reduzierte Expression opsonierender Moleküle wurde begleitet von einer veränderten proteasomalen Zusammensetzung in murinen Makrophagen und Lebergewebe. Zusammengefasst lässt sich sagen, dass diese Ergebnisse eine bisher unbekannte Rolle von Immunoproteasomalen Untereinheiten bei der Regulierung der angeborenen Immunantwort auf extrazelluläre bakterielle Infektionen unterstreichen. / Immunoproteasomes, harboring the active site subunits LMP7, LMP2, and MECL1 exert protective, regulatory or modulating functions during infection-induced immune responses. Immunoproteasomes are constitutively expressed in hematopoietic derived cells, constituting the first line of defense against invading pathogens. To clarify the impact of immunoproteasomes on the innate immune response against Streptococcus pneumoniae, we characterized the progression of disease and analyzed the local as well as systemic innate immune response in LMP7 ko mice by using a S. pneumoniae infection model. Data showed that mice deficient in LMP7 suffered from a more severe case of pneumonia which ended in a systemic inflammatory response indicated by aggravated clinical signs, diagnostic parameters, and immune suppression. The systemic inflammatory response probably established in consequence of an increased bacteremia and resulted in early mortality. Although, bacterial killing efficiency of LMP7 ko leukocytes was unaffected ex vivo, LMP7 ko mice exhibited a reduction in the transcription of genes encoding immune modulating molecules such as pentraxins, ficolins, and collectins, which facilitate opsonophagocytosis. The reduced expression of opsonins was accompanied by an affected subunit composition of proteasomes in murine macrophages and liver. In summary these results highlight an unsuspected role for immuno-subunits in modulating the innate immune response to extracellular bacterial infections.