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Abstract
Background
Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo‐epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram‐negative Escherichia coli.
Objectives
To assess the role of PF4, anti‐PF4/H antibodies and FcγRIIa in killing of Gram‐positive bacteria by platelets.
Methods
Binding of PF4 to protein‐A deficient Staphylococcus aureus (SA113Δspa) and non‐encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti‐PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P‐expression, PAC‐1 binding) and phosphatidylserine (PS)‐exposure were analyzed.
Results
Monoclonal and patient‐derived anti‐PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6‐fold increased fluorescence signal for human anti‐PF4/H antibodies to 24.0‐fold increase for KKO). Staphylococcus aureus (5.5 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/mL). Killing was not further enhanced by PF4 or anti‐PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP‐6 stimulation and exposed high levels of PS.
Conclusions
Anti‐PF4/H antibodies seem to have only a minor role for direct killing of Gram‐positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.
Exploring Virulence Factors and Alternative Therapies against Staphylococcus aureus Pneumonia
(2020)
Neue Antibiotika und Präventionsmaßnahmen gegen S. aureus sind aufgrund der starken Ausbreitung multiresistenter S. aureus-Stämme dringend erforderlich. Zur Entwicklung von Therapie- und Präventionsmaßnahmen werden geeignete Infektionsmodellen benötigt, die die klinische Situation möglichst exakt widerspiegeln. Da die Spezies S. aureus stark wirtsspezifisch ist, könnten wirtsadaptierte S. aureus-Stämme hierbei äußerst hilfreich sein. In der Infektionsforschung werden vor allem Mausmodelle verwendet. Da bisher jedoch angenommen wurde, dass Mäuse keine natürlichen Wirte von S. aureus sind, sind S. aureus-Forscher davon ausgegangen, dass Mäuse kein geeignetes Modell darstellen. Das wurde durch unsere und andere Arbeitsgruppen allerdings in den letzten Jahren widerlegt. Wir konnten zeigen, dass Labor- und Wildmäuse mit S. aureus besiedelt sind.
Im Rahmen dieser Arbeit sollte geklärt werden, ob murine Infektionsmodelle durch die Verwendung von mausadaptierten S. aureus-Stämmen optimiert werden können. Aus über 250 S. aureus-Stämmen, die aus Labor und Wildmäusen isoliert wurden, wurden vier mausadaptierte S. aureus-Isolate ausgewählt und mit dem humanen S. aureus-Isolat Newman in einem Pneumonie- und Bakteriämiemodell vergleichen. Diese Stämme wiesen einen repräsentativen spa-Typ sowie typischen Phagenmuster und Virulenzgene auf. Zudem waren sie in der Lage, murines Plasma zu koagulieren und in murinem Vollblut zu replizieren.
Es zeigte sich, dass das murine Isolat S. aureus DIP sowohl im Pneumonie- als auch im Bakteriämiemodell deutlich virulenter war als das humane Isolat Newman und die anderen getesteten mausadaptierten Stämme. Nach kürzester Zeit starben alle Tiere, die mit S. aureus DIP infiziert wurden. Wurde die Infektionsdosis im Vergleich zu Newman um 90 % reduziert, waren die bakterielle Last, der Belastungsscore, sowie die Zytokin- und Chemokinkonzentrationen nach Infektion mit S. aureus DIP bzw. S. aureus Newman vergleichbar. Im Besiedlungsmodell konnte gezeigt werden, dass die mausadaptierten Stämme S. aureus JSNZ sowie S. aureus DIP in der Lage sind, Mäuse über einen Zeitraum von 7 Tagen stabil zu besiedeln. Mäuse, die mit S. aureus Newman besiedelt waren, konnten den Stamm innerhalb dieses Zeitraums eliminieren. Die Genomsequenzierung der in vivo verwendeten S. aureus Stämme zeigte, dass lediglich S. aureus DIP für das Leukozidin LukMF‘ kodiert. Das lässt vermuten, dass die Präsenz des Virulenzfaktors für die gesteigerte Virulenz von S. aureus DIP verantwortlich sein könnte.
Des Weiteren sollten in dieser Arbeit ein Besiedlungsmodell mit murinen S. aureus-Isolaten etabliert und die beteiligten Immunzellen quantifiziert werden. Es zeigte sich, dass Mäuse mit murinen S. aureus-Isolaten bis zu 7 Tage besiedelt werden können wohingegen S. aureus Newman zu diesem Zeitpunkt nur noch in 20 % der Tiere nachweisbar war. Zudem konnte bei der intranasalen Besiedlung mit einer hohen Dosis S. aureus DIP [1 × 10^8 CFU] gezeigt werden, dass sowohl Th17-Zellen als auch γδ-T-Zellen nach 7 Tagen IL-17A, IL-17F und IL-22 produzieren. Jedoch konnte die Zytokinproduktion nur in Tieren nachgewiesen werden, die einen hohen Belastungsscore aufwiesen. Da nach 24 Stunden bei Tieren mit hohem Belastungsscore auch Bakterien in der Lunge detektiert wurde, ist anzunehmen, dass S. aureus diese Tiere nicht nur besiedelt, sondern bei ihnen auch eine Atemwegsinfektion verursacht hatte. Durch den geringen prozentualen Anteil an ILCs in den zervikalen Lymphknoten war es nicht möglich Rückschlüsse auf deren Zytokinproduktion zu ziehen. Somit gelang es zwar ein murines S. aureus-Besiedlungsmodell zu etablieren, jedoch kann keine Aussage zu den beteiligten Zellen des Immunsystems getroffen werden.
Zusammenfassend konnte gezeigt werden, dass Labormäuse mit mausadaptierten S. aureus-Stämmen länger besiedelt werden können als mit dem humanen Referenzstamm Newman. Zudem konnte mit Hilfe des mausadaptierten Stammes S. aureus DIP die Infektionsdosis im Pneumonie- und Bakteriämiemodell erheblich reduziert werden. Somit gelang es Mausmodelle durch die Verwendung von mausadaptierten S. aureus-Stämmen zu optimieren, auch wenn das nicht auf alle getesteten Isolate zutrifft. Durch die Anpassung an den murinen Wirt stellen mausadaptierte S. aureus-Stämme wie DIP und JSNZ ein physiologischeres Modell der Pathogen-Wirts-Interaktion dar. Die Verwendung eines solchen Stammes ermöglicht es ein besseres Verständnis für Infektionsprozesse und die Pathogen-Wirt-Interaktionen zu erlangen und dadurch eventuell neue Therapiemöglichkeiten zu entwickeln.
Es ist zu berücksichtigen, dass auch die Verwendung mausadaptierter S. aureus-Stämme in murinen Besiedlungs- und Infektionsmodellen lediglich ein Modell darstellt, welches Vor- und Nachteile hat. Daher ist es essenziell, dass Wissenschaftler die Grenzen jedes Modellsystems kennen und das richtige Infektionsmodell (oder eine Kombination davon) auswählen, um ihre Forschungsfragen zu beantworten.
Staphylococcus aureus (S. aureus) can secrete a broad range of virulence factors, among which staphylococcal serine protease-like proteins (Spls) have been identified as bacterial allergens. The S. aureus allergen serine protease-like protein D (SplD) induces allergic asthma in C57BL/6J mice through the IL-33/ST2 signaling axis. Analysis of C57BL/6J, C57BL/6N, CBA, DBA/2, and BALB/c mice treated with intratracheal applications of SplD allowed us to identify a frameshift mutation in the serine (or cysteine) peptidase inhibitor, clade A, and member 3I (Serpina3i) causing a truncated form of SERPINA3I in BALB/c, CBA, and DBA/2 mice. IL-33 is a key mediator of SplD-induced immunity and can be processed by proteases leading to its activation or degradation. Full-length SERPINA3I inhibits IL-33 degradation in vivo in the lungs of SplD-treated BALB/c mice and in vitro by direct inhibition of mMCP-4. Collectively, our results establish SERPINA3I as a regulator of IL-33 in the lungs following exposure to the bacterial allergen SplD, and that the asthma phenotypes of mouse strains may be strongly influenced by the observed frameshift mutation in Serpina3i. The analysis of this protease-serpin interaction network might help to identify predictive biomarkers for type-2 biased airway disease in individuals colonized by S. aureus.
Background: Annual transfusion rates in many European countries range between 25 and 35 red blood cell concentrates (RBCs)/1,000 population.It is unclear why transfusion rates in Germany are considerably higher (approx. 50–55 RBCs/1,000 population). Methods: We assessed the characteristics of transfusion recipients at all hospitals of the German federal state Mecklenburg-Western Pomerania during a 10-year longitudinal study. Results: Although 75% of patients received ≤4 RBCs/patient in 2015 (1 RBC: 11.3%; 2 RBCs: 42.6%; 3 RBCs: 6.3%; 4 RBCs: 15.0%), the mean transfusion index was 4.6 RBCs due to a minority of patients with a high transfusion demand. Two thirds of all RBCs were transfused to only 25% of RBC recipients. Consistently, male patients received a higher number of RBCs (2005: 54.2%; 2015: 56.8%) and had a higher mean transfusion index than female patients (mean 5.1 ± 7.2; median 2; inter-quartile range [IQR] 2–4 vs. mean 4.0 ± 5.8; median 2; IQR 2–4). The absolute transfusion demand decreased between 2005 and 2015 by 13.5% due to a composite of active reduction (clinical practice change) and population decline in the 65- to 75-year age group (lower birth rate cohort 1940–1950); however, with major differences between hospitals (range from –61.0 to +41.4%). Conclusion: Transfusion demand in a population could largely be driven by patients with high transfusion demand. Different treatment practices in this group of patients probably add to the major differences in transfusion demand per 1,000 individuals between countries. The available data cannot prove this hypothesis. Implementation of a diagnosis-related group-based monitoring system is urgently needed to allow informative monitoring on the population level and meaningful comparisons between transfusion practices.
Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO2) are the key enzymes of tryptophan (TRP) metabolism in the kynurenine pathway (KP). Both enzymes function as indicators of immunosuppression and poor survival in cancer patients. Direct or indirect targeting of either of these substances seems thus reasonable to improve therapy options for patients. In this study, glioblastoma multiforme (GBM) as well as head and neck squamous cell carcinomas (HNSCC) were examined because of their different mechanisms of spontaneous and treatment-induced immune escape. Effects on gene expression and protein levels were examined. Accompanying assessment of TRP metabolites from treated GBM cell culture supernatants was conducted. Our results show a heterogeneous and inversely correlated expression profile of TRP-metabolizing genes among GBM and HNSCC cells, with low, but inducible IDO1 expression upon IFNγ treatment. TDO2 expression was higher in GBM cells, while genes encoding kynurenine aminotransferases were mainly confined to HNSCC cells. These data indicate that the KP is active in both entities, with however different enzymes involved in TRP catabolism. Upon treatment with Temozolomide, the standard of care for GBM patients, IDO1 was upregulated. Comparable, although less pronounced effects were seen in HNSCC upon Cetuximab and conventional drugs (i.e., 5-fluorouracil, Gemcitabine). Here, IDO1 and additional genes of the KP (KYAT1, KYAT2, and KMO) were induced. Vice versa, the novel yet experimental cyclin-dependent kinase inhibitor Dinaciclib suppressed KP in both entities. Our comprehensive data imply inhibition of the TRP catabolism by Dinaciclib, while conventional chemotherapeutics tend to activate this pathway. These data point to limitations of conventional therapy and highlight the potential of targeted therapies to interfere with the cells' metabolism more than anticipated.
Infections are often caused by pathobionts, endogenous bacteria that belong to the microbiota. Trauma and surgical intervention can allow bacteria to overcome host defences, ultimately leading to sepsis if left untreated. One of the main defence strategies of the immune system is the production of highly specific antibodies. In the present proof-of-concept study, plasma antibodies against 9 major pathogens were measured in sepsis patients, as an example of severe systemic infections. The binding of plasma antibodies to bacterial extracellular proteins was quantified using a semi-automated immunoblot assay. Comparison of the pathogen-specific antibody levels before and after infection showed an increase in plasma IgG in 20 out of 37 tested patients. This host-directed approach extended the results of pathogen-oriented microbiological and PCR diagnostics: a specific antibody response to additional bacteria was frequently observed, indicating unrecognised poly-microbial invasion. This might explain some cases of failed, seemingly targeted antibiotic treatment.
Although antigen-specific priming of antibody responses is impaired during sepsis, there is nevertheless a strong increase in IgM and IgG serum concentrations. Using colon ascendens stent peritonitis (CASP), a mouse model of polymicrobial abdominal sepsis, we observed substantial increases in IgM as well as IgG of all subclasses, starting at day 3 and peaking 2 weeks after sepsis induction. The dominant source of antibody-secreting cells was by far the spleen, with a minor contribution of the mesenteric lymph nodes. Remarkably, sepsis induction in splenectomized mice did not change the dynamics of the serum IgM/IgG reaction, indicating that the marginal zone B cells, which almost exclusively reside in the spleen, are dispensable in such a setting. Hence, in systemic bacterial infection, the function of the spleen as dominant niche of antibody-producing cells can be compensated by extra-splenic B cell populations as well as other lymphoid organs. Depletion of CD4+ T cells did not affect the IgM response, while it impaired IgG generation of all subclasses with the exception of IgG3. Taken together, our data demonstrate that the robust class-switched antibody response in sepsis encompasses both T cell-dependent and -independent components.