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.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

The innate immune protein surfactant protein (SP-) D is a carbohydrate binding protein that was originally isolated from mucosal lung tissues. Recently, studies show that SP-D binds to antibodies, including immunoglobulin M (IgM), which interacts with late apoptotic cells. Here we focus on the interaction between SP-D and IgM as they pertain to late apoptotic cell clearance. We hypothesized that the three-way interaction between IgM, SP-D and late apoptotic cells is functionally applicable to clearing late apoptotic cells from the lungs, thereby reducing lung inflammation. We show that SP-D binds to IgM and that IgM binds to the late apoptotic subclass of dying cells. We demonstrate that IgM and SP-D can both bind to late apoptotic cells in mutually distinct regions while also displaying some regional overlap. We show evidence that during LPS-induced lung inflammation both IgM and SP-D levels are elevated and this corresponds to an augmentation of apoptotic cell clearance. We illustrate that the protein interaction of IgM and SP-D is functionally relevant to apoptotic cell clearance in the lungs by showing that late apoptotic cells coated in IgM and/or SP-D are cleared more efficiently than control cells, by alveolar macrophages in vivo. Our ex vivo studies further show that these cells internalize apoptotic cells by engulfing very small particles released from the dying cells. We then showed that IgM preferentially directs the engulfment of small particles (~1 μm) by macrophages, in an apparent size-specific antibody-dependent particle clearance function. Our data reveals a novel relationship amongst IgM, SP-D, apoptotic cells, and alveolar macrophages that contributes to our understanding of apoptotic cell clearance, which may be used in the future to generate strategies addressing apoptotic cell accumulation or clearance deficiency in disease.

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.