Myogenin controls via AKAP6 non-centrosomal microtubule-organizing center formation at the nuclear envelope

Non-centrosomal microtubule-organizing centers (MTOCs) are pivotal for the function of multiple cell types, but the processes initiating their formation are unknown. Here, we find that the transcription factor myogenin is required in murine myoblasts for the localization of MTOC proteins to the nuclear envelope. Moreover, myogenin is sufficient in fibroblasts for nuclear envelope MTOC (NE-MTOC) formation and centrosome attenuation. Bioinformatics combined with loss- and gain-of-function experiments identified induction of AKAP6 expression as one central mechanism for myogenin-mediated NE-MTOC formation. Promoter studies indicate that myogenin preferentially induces the transcription of muscle- and NE-MTOC-specific isoforms of Akap6 and Syne1, which encodes nesprin-1α, the NE-MTOC anchor protein in muscle cells. Overexpression of AKAP6β and nesprin-1α was sufficient to recruit endogenous MTOC proteins to the nuclear envelope of myoblasts in the absence of myogenin. Taken together, our results illuminate how mammals transcriptionally control the switch from a centrosomal MTOC to an NE-MTOC and identify AKAP6 as a novel NE-MTOC component in muscle cells

AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9

The switch from centrosomal microtubule-organizing centers (MTOCs) to non-centrosomal MTOCs during differentiation is poorly understood. Here, we identify AKAP6 as key component of the nuclear envelope MTOC. In rat cardiomyocytes, AKAP6 anchors centrosomal proteins to the nuclear envelope through its spectrin repeats, acting as an adaptor between nesprin-1α and Pcnt or AKAP9. In addition, AKAP6 and AKAP9 form a protein platform tethering the Golgi to the nucleus. Both Golgi and nuclear envelope exhibit MTOC activity utilizing either AKAP9, or Pcnt-AKAP9, respectively. AKAP6 is also required for formation and activity of the nuclear envelope MTOC in human osteoclasts. Moreover, ectopic expression of AKAP6 in epithelial cells is sufficient to recruit endogenous centrosomal proteins. Finally, AKAP6 is required for cardiomyocyte hypertrophy and osteoclast bone resorption activity. Collectively, we decipher the MTOC at the nuclear envelope as a bi-layered structure generating two pools of microtubules with AKAP6 as a key organizer

Genomic investigations of unexplained acute hepatitis in children

Since its first identification in Scotland, over 1,000 cases of unexplained paediatric hepatitis in children have been reported worldwide, including 278 cases in the UK1. Here we report an investigation of 38 cases, 66 age-matched immunocompetent controls and 21 immunocompromised comparator participants, using a combination of genomic, transcriptomic, proteomic and immunohistochemical methods. We detected high levels of adeno-associated virus 2 (AAV2) DNA in the liver, blood, plasma or stool from 27 of 28 cases. We found low levels of adenovirus (HAdV) and human herpesvirus 6B (HHV-6B) in 23 of 31 and 16 of 23, respectively, of the cases tested. By contrast, AAV2 was infrequently detected and at low titre in the blood or the liver from control children with HAdV, even when profoundly immunosuppressed. AAV2, HAdV and HHV-6 phylogeny excluded the emergence of novel strains in cases. Histological analyses of explanted livers showed enrichment for T cells and B lineage cells. Proteomic comparison of liver tissue from cases and healthy controls identified increased expression of HLA class 2, immunoglobulin variable regions and complement proteins. HAdV and AAV2 proteins were not detected in the livers. Instead, we identified AAV2 DNA complexes reflecting both HAdV-mediated and HHV-6B-mediated replication. We hypothesize that high levels of abnormal AAV2 replication products aided by HAdV and, in severe cases, HHV-6B may have triggered immune-mediated hepatic disease in genetically and immunologically predisposed children

Systematic investigation of the interplay between biomaterials and the immune system in vitro

Die Nutzung medizinischer Implantate ist heutzutage weit verbreitet. Unmittelbar nach Gewebekontakt haften Proteine an der Oberfläche des Implantats an und es kommt zu einer Aktivierung der umgebenden Zellen, was zu einer initialen Entzündungsreaktion führt. Entwickelt sich diese Reaktion zu einem chronischen Entzündungszustand, können erhebliche negative Auswirkungen wie die Abstoßung und der Verlust des Implantats auftreten. Das Ausmaß der Immunreaktion wird dabei stark von den physikalisch-chemischen Eigenschaften der verwendeten Biomaterialien beeinflusst. Daher kann die Modulation von Oberflächeneigenschaften wie Topographie oder Benetzbarkeit eine praktikable Strategie zur Veränderung der Immunreaktion sein. Die komplexe Beziehung zwischen den Oberflächen-eigenschaften, der hochsensiblen Adsorption von Proteinen, und der vielschichtigen Immunantwort ist jedoch noch weitgehend ungelöst. Ziel dieser Arbeit ist es, das Zusammenspiel dieser drei kritischen Faktoren, welche die Biokompatibilität eines Implantats bestimmen, zu untersuchen, um eine wirksame Modulation der Immunantwort auf biomedizinische Implantate zu ermöglichen. Im ersten Teil dieser Arbeit wurde die Rolle der verschiedenen Immunzellarten untersucht, wobei Monozyten/Makrophagen als wesentliche Vermittler der initialen Entzündungsreaktion identifiziert wurden, während die Beteiligung von T- und NK-Zellen in dieser Phase nur gering war. Die Untersuchung von Zahnimplantatproben aus Titan offenbarte oberflächenabhängige Immunreaktionen in Abhängigkeit von Benetzbarkeit und Rauheit. Im Gegensatz dazu zeigte die systematische Analyse des Einflusses der Oberflächenrauheit von Polymermaterialien im zweiten Teil der Arbeit eine ähnliche immunologische Aktivierung, unabhängig von der untersuchten Oberflächenrauheit im gesamten Testbereich. Diese war unabhängig von der biologischen Komplexität des verwendeten Zellkultursystems (Makrophagenzelllinie, PBMCs, Vollblut). Der letzte Teil der Arbeit untersuchte die durch die Benetzbarkeit vermittelten Auswirkungen auf die Aktivität von Immunzellen unter Verwendung von PEM Beschichtungen und stellte fest, dass pro- und anti-inflammatorische Zytokinreaktionen in hohem Maße von der Benetzbarkeit abhängig waren, wobei die pro-inflammatorischen Effekte bei Oberflächen mit größerer Hydrophilie geringer ausfielen. Experimente mit serumfreiem Zellkulturmedium zeigten, dass die beobachteten Effekte eindeutig von der Anwesenheit von Serumproteinen auf der Biomaterialoberfläche abhängig sind. Mittels massenspektrometrischer Analyse wurden signifikante Veränderungen in der Art und Menge der adsorbierten Proteine festgestellt. Die beobachteten immunologischen Unterschiede konnten mit dem Vorhandensein spezifischer Apolipoproteine auf den Oberflächen korreliert werden, was darauf hindeutet, dass Apolipoproteine eine wichtige Rolle bei der Modulation der Immunantwort von Biomaterialien spielen könnten. Diese Ergebnisse könnten die gezielte Konzipierung immunmodulatorischer Oberflächen unterstützen, um die Heilung und Implantatintegration zu fördern. Darüber hinaus legen sie einen größeren Schwerpunkt auf die Adsorption von Proteinen wie Apolipoproteinen als entscheidende Klasse von Immunzellmediatoren.Medical implants are widely used nowadays. Following tissue contact, proteins adhere to the device’s surface and surrounding cells become activated, causing an initial inflammatory host response. If this host response develops into a chronic inflammatory state, significant adverse effects such as implant rejection and loss might occur. The physico-chemical qualities of the biomaterials employed have a substantial influence on the degree of the immune response. Therefore, modulating surface properties such as topography or wettability may be a viable strategy for altering the immune response. However, the complex interrelation between surface properties, highly sensitive adsorption of proteins, and multifaceted immune response remains largely unresolved. This thesis aims at investigating the interplay of all three of these critical factors determining a device’s biocompatibility in order to enable effective modulation of the immune response to biomedical implants. In the first part of this thesis, investigation of the contributing role of different immune cell types identified monocytes/macrophages as essential mediators of the initial inflammatory response, while involvement of T and NK cells was just minor during this phase. Analysis of titanium dental implant specimen revealed surface-dependent immune responses related to wettability and roughness. In contrast, systematic analysis of the influence of surface roughness of polymer materials in the second part of the thesis showed similar immunological activation irrespective of the applied surface roughness throughout the tested range. This was independent of the biological complexity of the cell culture system used (macrophage cell line, PBMCs, whole blood). The final section of the thesis examined wettability-mediated effects on immune cell activity using PEM coatings and discovered that pro- and anti-inflammatory cytokine responses are highly dependent on wettability, with lower pro-inflammatory effects reported on the more hydrophilic PEM surface. Experiments using serum-free cell culture medium demonstrated that the observed effects are clearly dependent on the presence of serum proteins at the biomaterial surface. Significant changes in the type and amount of adsorbed proteins were discovered using mass spectrometry analysis. The observed immunological differences could be correlated with the presence of specific apolipoproteins at the surfaces, implying that apolipoproteins might play a significant role in the modulation of biomaterial immune responses. These findings may aid in the targeted design of immunomodulatory surfaces to promote healing and implant integration. In addition, they place a larger emphasis on adsorption of proteins such as apolipoproteins as crucial class of immune cell mediators

Systematic investigation of the interplay between biomaterials and the immune system in vitro

Die Nutzung medizinischer Implantate ist heutzutage weit verbreitet. Unmittelbar nach Gewebekontakt haften Proteine an der Oberfläche des Implantats an und es kommt zu einer Aktivierung der umgebenden Zellen, was zu einer initialen Entzündungsreaktion führt. Entwickelt sich diese Reaktion zu einem chronischen Entzündungszustand, können erhebliche negative Auswirkungen wie die Abstoßung und der Verlust des Implantats auftreten. Das Ausmaß der Immunreaktion wird dabei stark von den physikalisch-chemischen Eigenschaften der verwendeten Biomaterialien beeinflusst. Daher kann die Modulation von Oberflächeneigenschaften wie Topographie oder Benetzbarkeit eine praktikable Strategie zur Veränderung der Immunreaktion sein. Die komplexe Beziehung zwischen den Oberflächen-eigenschaften, der hochsensiblen Adsorption von Proteinen, und der vielschichtigen Immunantwort ist jedoch noch weitgehend ungelöst. Ziel dieser Arbeit ist es, das Zusammenspiel dieser drei kritischen Faktoren, welche die Biokompatibilität eines Implantats bestimmen, zu untersuchen, um eine wirksame Modulation der Immunantwort auf biomedizinische Implantate zu ermöglichen. Im ersten Teil dieser Arbeit wurde die Rolle der verschiedenen Immunzellarten untersucht, wobei Monozyten/Makrophagen als wesentliche Vermittler der initialen Entzündungsreaktion identifiziert wurden, während die Beteiligung von T- und NK-Zellen in dieser Phase nur gering war. Die Untersuchung von Zahnimplantatproben aus Titan offenbarte oberflächenabhängige Immunreaktionen in Abhängigkeit von Benetzbarkeit und Rauheit. Im Gegensatz dazu zeigte die systematische Analyse des Einflusses der Oberflächenrauheit von Polymermaterialien im zweiten Teil der Arbeit eine ähnliche immunologische Aktivierung, unabhängig von der untersuchten Oberflächenrauheit im gesamten Testbereich. Diese war unabhängig von der biologischen Komplexität des verwendeten Zellkultursystems (Makrophagenzelllinie, PBMCs, Vollblut). Der letzte Teil der Arbeit untersuchte die durch die Benetzbarkeit vermittelten Auswirkungen auf die Aktivität von Immunzellen unter Verwendung von PEM Beschichtungen und stellte fest, dass pro- und anti-inflammatorische Zytokinreaktionen in hohem Maße von der Benetzbarkeit abhängig waren, wobei die pro-inflammatorischen Effekte bei Oberflächen mit größerer Hydrophilie geringer ausfielen. Experimente mit serumfreiem Zellkulturmedium zeigten, dass die beobachteten Effekte eindeutig von der Anwesenheit von Serumproteinen auf der Biomaterialoberfläche abhängig sind. Mittels massenspektrometrischer Analyse wurden signifikante Veränderungen in der Art und Menge der adsorbierten Proteine festgestellt. Die beobachteten immunologischen Unterschiede konnten mit dem Vorhandensein spezifischer Apolipoproteine auf den Oberflächen korreliert werden, was darauf hindeutet, dass Apolipoproteine eine wichtige Rolle bei der Modulation der Immunantwort von Biomaterialien spielen könnten. Diese Ergebnisse könnten die gezielte Konzipierung immunmodulatorischer Oberflächen unterstützen, um die Heilung und Implantatintegration zu fördern. Darüber hinaus legen sie einen größeren Schwerpunkt auf die Adsorption von Proteinen wie Apolipoproteinen als entscheidende Klasse von Immunzellmediatoren.Medical implants are widely used nowadays. Following tissue contact, proteins adhere to the device’s surface and surrounding cells become activated, causing an initial inflammatory host response. If this host response develops into a chronic inflammatory state, significant adverse effects such as implant rejection and loss might occur. The physico-chemical qualities of the biomaterials employed have a substantial influence on the degree of the immune response. Therefore, modulating surface properties such as topography or wettability may be a viable strategy for altering the immune response. However, the complex interrelation between surface properties, highly sensitive adsorption of proteins, and multifaceted immune response remains largely unresolved. This thesis aims at investigating the interplay of all three of these critical factors determining a device’s biocompatibility in order to enable effective modulation of the immune response to biomedical implants. In the first part of this thesis, investigation of the contributing role of different immune cell types identified monocytes/macrophages as essential mediators of the initial inflammatory response, while involvement of T and NK cells was just minor during this phase. Analysis of titanium dental implant specimen revealed surface-dependent immune responses related to wettability and roughness. In contrast, systematic analysis of the influence of surface roughness of polymer materials in the second part of the thesis showed similar immunological activation irrespective of the applied surface roughness throughout the tested range. This was independent of the biological complexity of the cell culture system used (macrophage cell line, PBMCs, whole blood). The final section of the thesis examined wettability-mediated effects on immune cell activity using PEM coatings and discovered that pro- and anti-inflammatory cytokine responses are highly dependent on wettability, with lower pro-inflammatory effects reported on the more hydrophilic PEM surface. Experiments using serum-free cell culture medium demonstrated that the observed effects are clearly dependent on the presence of serum proteins at the biomaterial surface. Significant changes in the type and amount of adsorbed proteins were discovered using mass spectrometry analysis. The observed immunological differences could be correlated with the presence of specific apolipoproteins at the surfaces, implying that apolipoproteins might play a significant role in the modulation of biomaterial immune responses. These findings may aid in the targeted design of immunomodulatory surfaces to promote healing and implant integration. In addition, they place a larger emphasis on adsorption of proteins such as apolipoproteins as crucial class of immune cell mediators

Raman Microspectroscopy Identifies Biochemical Activation Fingerprints in THP-1- and PBMC-Derived Macrophages

(1) The monocytic leukemia cell line THP-1 and primary monocyte-derived macrophages (MDMs) are popular in vitro model systems to study human innate immunity, wound healing, and tissue regeneration. However, both cell types differ significantly in their origin and response to activation stimuli. (2) Resting THP-1 and MDMs were stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ) and analyzed by Raman microspectroscopy (RM) before and 48 h after activation. Raman data were subsequently analyzed using principal component analysis. (3) We were able to resolve and analyze the spatial distribution and molecular composition of proteins, nucleic acids, and lipids in resting and activated THP-1 and MDMs. Our findings reveal that proinflammatory activation-induced significant spectral alterations at protein and phospholipid levels in THP-1. In MDMs, we identified that nucleic acid and non-membrane-associated intracellular lipid composition were also affected. (4) Our results show that it is crucial to carefully choose the right cell type for an in vitro model as the nature of the cells itself may impact immune cell polarization or activation results. Moreover, we demonstrated that RM is a sensitive tool for investigating cell-specific responses to activation stimuli and monitoring molecular changes in subcellular structures

Raman Microspectroscopy Identifies Biochemical Activation Fingerprints in THP-1- and PBMC-Derived Macrophages

(1) The monocytic leukemia cell line THP-1 and primary monocyte-derived macrophages (MDMs) are popular in vitro model systems to study human innate immunity, wound healing, and tissue regeneration. However, both cell types differ significantly in their origin and response to activation stimuli. (2) Resting THP-1 and MDMs were stimulated with lipopolysaccharide (LPS) and interferon γ (IFNγ) and analyzed by Raman microspectroscopy (RM) before and 48 h after activation. Raman data were subsequently analyzed using principal component analysis. (3) We were able to resolve and analyze the spatial distribution and molecular composition of proteins, nucleic acids, and lipids in resting and activated THP-1 and MDMs. Our findings reveal that proinflammatory activation-induced significant spectral alterations at protein and phospholipid levels in THP-1. In MDMs, we identified that nucleic acid and non-membrane-associated intracellular lipid composition were also affected. (4) Our results show that it is crucial to carefully choose the right cell type for an in vitro model as the nature of the cells itself may impact immune cell polarization or activation results. Moreover, we demonstrated that RM is a sensitive tool for investigating cell-specific responses to activation stimuli and monitoring molecular changes in subcellular structures

Lipidome profiling with Raman microspectroscopy identifies macrophage response to surface topographies of implant materials

Biomaterial characteristics such as surface topographies have been shown to modulate macrophage phenotypes. The standard methodologies to measure macrophage response to biomaterials are marker-based and invasive. Raman microspectroscopy (RM) is a marker-independent, noninvasive technology that allows the analysis of living cells without the need for staining or processing. In the present study, we analyzed human monocyte-derived macrophages (MDMs) using RM, revealing that macrophage activation by lipopolysaccharides (LPS), interferons (IFN), or cytokines can be identified by lipid composition, which significantly differs in M0 (resting), M1 (IFN-γ/LPS), M2a (IL-4/IL-13), and M2c (IL-10) MDMs. To identify the impact of a biomaterial on MDM phenotype and polarization, we cultured macrophages on titanium disks with varying surface topographies and analyzed the adherent MDMs with RM. We detected surface topography–induced changes in MDM biochemistry and lipid composition that were not shown by less sensitive standard methods such as cytokine expression or surface antigen analysis. Our data suggest that RM may enable a more precise classification of macrophage activation and biomaterial–macrophage interaction

Altered proinflammatory responses to polyelectrolyte multilayer coatings are associated with differences in protein adsorption and wettability

A full understanding of the relationship between surface properties, protein adsorption, and immune responses is lacking but is of great interest for the design of biomaterials with desired biological profiles. In this study, polyelectrolyte multilayer (PEM) coatings with gradient changes in surface wettability were developed to shed light on how this impacts protein adsorption and immune response in the context of material biocompatibility. The analysis of immune responses by peripheral blood mononuclear cells to PEM coatings revealed an increased expression of proinflammatory cytokines tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 and the surface marker CD86 in response to the most hydrophobic coating, whereas the most hydrophilic coating resulted in a comparatively mild immune response. These findings were subsequently confirmed in a cohort of 24 donors. Cytokines were produced predominantly by monocytes with a peak after 24 h. Experiments conducted in the absence of serum indicated a contributing role of the adsorbed protein layer in the observed immune response. Mass spectrometry analysis revealed distinct protein adsorption patterns, with more inflammation-related proteins (e.g., apolipoprotein A-II) present on the most hydrophobic PEM surface, while the most abundant protein on the hydrophilic PEM (apolipoprotein A-I) was related to anti-inflammatory roles. The pathway analysis revealed alterations in the mitogen-activated protein kinase (MAPK)-signaling pathway between the most hydrophilic and the most hydrophobic coating. The results show that the acute proinflammatory response to the more hydrophobic PEM surface is associated with the adsorption of inflammation-related proteins. Thus, this study provides insights into the interplay between material wettability, protein adsorption, and inflammatory response and may act as a basis for the rational design of biomaterials