Krüppel-like Faktor 4 als Regulator der Inflammation in der Pneumokokkenpneumonie

Streptococcus pneumoniae ist der Haupterreger der ambulant erworbenen Pneumonie, welche jährlich weltweit geschätzte 1,2 Millionen Todesopfer fordert und ein erhebliches medizinisches und sozioökonomisches Problem darstellt. Steigende Resistenzraten der Pneumokokken gegen wirksame Antibiotika und ein bislang unzureichender Impfschutz machen die Entschlüsselung der Interaktionsmechanismen zwischen Pneumokokken und Zellen des angeborenen Immunsystems zur Erschließung neuer Therapiemöglichkeiten unabdingbar. Die Aktivierung myeloider Zellen im alveolären Kompartiment ist von entscheidender Wichtigkeit für die Eliminierung eindringender Pathogene in die Lunge. Die Ausgangshypothese der vorliegenden Dissertation lautet, dass der Stammzellfaktor Krüppel- like Faktor 4 (KLF4) für die Aktivierung von Makrophagen durch Streptococcus pneumoniae eine wichtige Rolle spielt. Dabei konnte gezeigt werden, dass Pneumokokken dosisabhängig KLF4 in murinen, aus Knochenmarkszellen differenzierten Makrophagen (BMMs) induzieren. Diese Induktion erfolgt nur durch lebende Pneumokokken, die direkten Kontakt mit den BMMs haben und Autolysin LytA-abhängig DNA freisetzen. LytA-defiziente Pneumokokken können KLF4 in BMMs nicht induzieren. Werden den LytA-defizienten Pneumokokken freie prokaryotische oder fremde (humane) bzw. eigene (murine) eukaryotische DNA-Moleküle zugesetzt, wird die KLF4 Induktion wiederhergestellt. Experimente mit TLR9, MyD88 und TRIF knockout BMMs zeigten, dass TLR9, MyD88 und TRIF teilweise an dieser Induktion beteiligt sind. Zur Untersuchung der Funktion von KLF4 wurden BMMs aus in vivo mit 4-Hydroxytamoxifen (4-OHT) behandelten ERT-Cre+/- /KLF4loxP/loxP (KLF4 knockdown) Mäusen eingesetzt. Mit Pneumokokken stimulierte KLF4 knockdown BMMs bildeten weniger Keratinozyten Chemokin (engl. „keratinocyte chemoattractant“, KC) und mehr Interleukin (IL) 10 als die entsprechenden Wildtypen. Zusammenfassend konnte im Rahmen der Dissertation gezeigt werden, dass Pneumokokken KLF4 in BMMs über TLR9, MyD88, TRIF sowie einen bisher unbekannten DNA-Rezeptor induzieren, was zur Ausbildung eines proinflammatorischen Makrophagenphaenotyps führt.Streptococcus pneumoniae, or pneumococcus, is the agent most prone to cause community- acquired pneumonia (CAP), resulting in 1.2 million deaths every year worldwide and therefore representing a major medical and socio-economical challenge. The increasing resistance of the pneumococci against effective antibiotics and the insufficient protection provided by vaccination underline the necessity to comprehend the interaction between the pneumococci and the innate immune cells in order to develop new therapeutic strategies. The activation of myeloid cells in the alveolar compartment is of utmost importance for eliminating invading pathogens in the lungs. The hypothesis of this thesis is that the transcription factor Krueppel-like factor 4 (KLF4) plays an essential role in the activation of macrophages by pneumococci. The results of the current analysis have shown that the pneumococci induce a dose-dependent KLF4 expression in murine bone marrow-derived macrophages (BMMs). However, only viable pneumococci, which have direct contact to the host cells and release autolysin LytA-dependent DNA, induce KLF4. Mutants lacking autolysin LytA were not able to induce KLF4. Yet, the exogenous supplementation of prokaryotic and foreign (human) or own (mouse) eukaryotic DNA restored pneumococci- related induction of KLF4 by these mutant bacteria. Experiments using TLR9, MyD88 and TRIF knockout BMMs revealed that TLR9, MyD88 and TRIF were partly involved in the induction of KLF4. In order to investigate the function of KLF4, BMMs of in vivo with 4- hydroxytamoxifen (4-OHT) induced ERT-Cre+/-/KLF4loxP/loxP (KLF4 knockdown) mice were used. The loss of KLF4 expression reduced the pneumococci-dependent release of keratinocyte chemoattractant (KC) and enhanced the secretion of interleukin (IL) 10. To conclude, pneumococci-related KLF4 induction in BMMs is partly mediated via TLR9, MyD88, TRIF and a hitherto unknown host cell DNA sensor leading to a proinflammatory macrophage phenotype

Induction of Krüppel-Like Factor 4 Mediates Polymorphonuclear Neutrophil Activation in Streptococcus pneumoniae Infection

The recruitment and activation of polymorphonuclear neutrophils (PMNs) are of central importance for the elimination of pathogens in bacterial infections. We investigated the Streptococcus pneumoniae-dependent induction of the transcription factor Kruppel-like factor (KLF) 4 in PMNs as a potential regulator of PMN activation. We found that KLF4 expression is induced in human blood-derived PMNs in a time- and dose-dependent manner by wild-type S. pneumoniae and capsule knockout mutants. Unencapsulated knockout mutants induced stronger KLF4 expression than encapsulated wild types. The presence of autolysin LytA-competent (thus viable) pneumococci and LytA-mediated bacterial autolysis were required for KLF4 induction in human and murine PMNs. LyzMcre-mediated knockdown of KLF4 in murine blood-derived PMNs revealed that KLF4 influences pneumococci killing and increases the release of the proinflammatory cytokines tumor necrosis factor alpha and keratinocyte chemoattractant and decreases the release of the anti-inflammatory cytokine interleukin-10. Thus, S. pneumoniae induces KLF4 expression in PMNs, which contributes to PMN activation in S. pneumoniae infection

Krueppel-Like Factor 4 Expression in Phagocytes Regulates Early Inflammatory Response and Disease Severity in Pneumococcal Pneumonia

The transcription factor Krueppel-like factor (KLF) 4 fosters the pro-inflammatory immune response in macrophages and polymorphonuclear neutrophils (PMNs) when stimulated with Streptococcus pneumoniae, the main causative pathogen of community-acquired pneumonia (CAP). Here, we investigated the impact of KLF4 expression in myeloid cells such as macrophages and PMNs on inflammatory response and disease severity in a pneumococcal pneumonia mouse model and in patients admitted to hospital with CAP. We found that mice with a myeloid-specific knockout of KLF4 mount an insufficient early immune response with reduced levels of pro-inflammatory cytokines and increased levels of the anti-inflammatory cytokine interleukin (IL) 10 in bronchoalveolar lavage fluid and plasma and an impaired bacterial clearance from the lungs 24 hours after infection with S. pneumoniae. This results in higher rates of bacteremia, increased lung tissue damage, more severe symptoms of infection and reduced survival. Higher KLF4 gene expression levels in the peripheral blood of patients with CAP at hospital admission correlate with a favourable clinical presentation (lower sequential organ failure assessment (SOFA) score), lower serum levels of IL-10 at admission, shorter hospital stay and lower mortality or requirement of intensive care unit treatment within 28 days after admission. Thus, KLF4 in myeloid cells such as macrophages and PMNs is an important regulator of the early proinflammatory immune response and, therefore, a potentially interesting target for therapeutic interventions in pneumococcal pneumonia

Ascites’ neutrophil function is significantly impaired in patients with decompensated cirrhosis but can be restored by autologous plasma incubation

Systemic immune cell dysfunction is a typical feature of liver diseases and increases the risk of bacterial infection, especially spontaneous bacterial peritonitis. We evaluated functional properties of neutrophil granulocytes in blood and ascites of patients both with and without decompensated cirrhosis. We collected blood and ascites samples from 63 patients with cirrhosis and eight without cirrhosis. Phagocytosis activity (PA) and oxidative burst activity (OBA) were evaluated after ex vivo stimulation with E. coli, while fluorescence signals were measured by flow cytometry. Ascites’ neutrophil function tests were repeated after incubation with autologous plasma. Ascites’ neutrophils showed an impaired PA and OBA (median blood PA 98.1% (86.8–99.8) vs. ascites’ PA 50.5% (0.4–97.3), p < 0.0001; median blood OBA 98.7% (27.5–100) vs. ascites’ OBA 27.5% (0.3–96.7), p < 0.0001). Patients with non- cirrhotic ascites showed higher PA but equally suppressed OBA. Ascites’ neutrophil function could be partially restored after incubation with autologous plasma (median increase PA: 22.5% (−49.7 – +93.2), p = 0.002; OBA: 22.8% (−10.4 – +48.8), p = 0.002). Ascites’ neutrophils of patients with cirrhosis are functionally impaired, but could be partially restored after incubation with plasma. Further investigations are needed to identify the factors in ascites that are associated with neutrophils’ function

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

COVID-19 and the liver – Lessons learned

Liver involvement, indicated by elevated liver function test results, is common in hospitalized patients with coronavirus disease 2019 (COVID-19) and has been linked to disease severity and outcome. A dual pattern of elevated liver function tests can be observed especially in patients with severe or critical COVID-19, characterized by an increase in aminotransferases early in the course of this disease, followed by an increase in cholestasis-associated biochemistry markers at later stages. This dual pattern is associated with inflammatory response markers and poor outcome. Current notions on the mechanisms of liver injury in COVID-19 include direct cytopathic effects of the virus on hepatocytes and cholangiocytes, ischemic and hypoxic liver damage, drug-induced liver injury, activation of hepatic immune cells by excess cytokine production and exacerbation of pre-existing liver disease. Patients with obesity-related non-alcoholic fatty liver disease and, in particular, patients with cirrhosis are at high risk of liver injury and a fatal outcome from COVID-19. In contrast, individuals receiving stable immunosuppressive medication for autoimmune liver diseases or during long-term follow-up after liver transplantation do not have a higher case-to-infection ratio and have a fairly favourable outcome. The present review describes the epidemiology, characteristics and potential pathological mechanisms of COVID-19-related liver injury. Moreover, the influence of pre-existing liver disease on the susceptibility and severity of liver injury in COVID-19 are discussed

DNA-release by Streptococcus pneumoniae autolysin LytA induced Krueppel-like factor 4 expression in macrophages

14 p.-8 fig.The recruitment of myeloid cells to the lung is of utmost importance for the elimination of invading pathogens. We investigated the Streptococcus pneumoniae-dependent induction mechanism of KLF4 in macrophages as a potential regulator of the macrophage immune response. We demonstrated that only viable pneumococci, which have direct contact to the host cells and release LytA-dependent DNA, induced KLF4. Exogenous supplementation of pneumococcal, other bacterial, eukaryotic foreign (human) or self (mouse) DNA to autolysis-deficient pneumococci restored (at least in part) pneumococci-related KLF4 induction. Experiments using TLR9, TRIF and MyD88 knockout macrophages revealed that TLR9, TRIF and MyD88 were partly involved in the S. pneumoniae-induced KLF4 expression. BMMs missing important DNA receptor related molecules (ASC-/-, STING-/-) showed no differences in pneumococci-related KLF4 expression. Similar results were observed with IFNAR-/- BMMs and Type I IFN stimulated cells. LyzMcre mediated knockdown of KLF4 in BMMs resulted in a decreased secretion of proinflammatory cytokines and enhanced IL-10 release. In summary, we showed that pneumococci-related KLF4 induction in macrophages is mediated via a PAMP-DAMP induction mechanism involving a hitherto unknown host cell DNA sensor leading to a more proinflammatory macrophage phenotype.This work was supported by DFG SFB-TR84 grants to J. Zahlten, S. Hippenstiel and N. Suttorp (Projects C2, B6, B1), Jürgen Manchot Stiftung to T. Herta, A. Bhattacharyya and C. Kabus, SAF2012-39444-C02-01 and CIBER de Enfermedades Respiratorias (an initiative of the Instituto de Salud Carlos III (ISCIII)) to P. García.Peer reviewe