MSC besitzen vielfältige immunmodulatorische Eigenschaften, die sowohl über die Sekretion parakriner Signalmoleküle, wie Zytokine oder Wachstumsfaktoren, als auch über direkte Zell-Zell-Kontakte vermittelt werden. Durch Präkonditionierung, z.B. mit spezifischen Zytokinen, können die immunmodulatorischen Eigenschaften von MSC beeinflusst werden. Eine solche zielgerichtete Modifikation scheint insbesondere für therapeutische Anwendungen interessant. Eine Herausforderung auf dem Gebiet der MSC-Forschung ist die Diskrepanz zwischen vielversprechenden in vitro- und teilweise enttäuschenden in vivo-Ergebnissen. Für das ambitionierte Ziel eines breiten therapeutischen Einsatzes von MSC sind daher noch weitreichende Forschungen zum besseren Verständnis insbesondere der Interaktionen von MSC mit Zellen des Immunsystems notwendig. Ziel dieser Studie war es, Erkenntnisse über mögliche Prozesse zu gewinnen, die im Rahmen der Wundheilung bei Exposition von MSC mit proinflammatorischen Stimuli ablaufen. Im lokalen postoperativen Milieu treffen verschiedene proinflammatorische Faktoren sowie MSC und Zellen des Immunsystems aufeinander. Es wurde untersucht, ob die Präkonditionierung von MSC mit WS von Patienten nach tumorchirurgischen Eingriffen einen Einfluss auf das immunmodulatorische Potential hat. Hierbei konnte eine signifikante Erhöhung des immunsuppressiven Charakters von MSC unter WS-Präkonditionierung festgestellt werden. Gerade in unterschiedlichen immunologischen Milieus weisen MSC ein hohes Maß an Heterogenität in ihren Eigenschaften auf. Zukünftige Arbeiten sollten daher den Einfluss weiterer immunologischer Faktoren sowohl unter pro- als auch antiinflammatorischen Stimuli auf die durch MSC vermittelte Immunmodulation untersuchen. So können neue Möglichkeiten für eine effektive Präkonditionierung identifiziert werden, um die parakrine Variabilität in präklinischen und klinischen Studien zu minimieren und letztlich die therapeutische Wirksamkeit von MSC zu optimieren.

The spore-forming opportunistic enteropathogen Clostridioides difficile (C. difficile) has emerged as the most common cause of nosocomial antibiotic-associated diarrhea. Disruption of the healthy gut microbiota upon antibiotic therapy is the major risk factor for developing an infection. The symptoms can range from mild diarrhea to life threatening disease, such as colitis and tissue necrosis. The severity of infection is mediated by the expression of several virulence factors, including two major exotoxins. Many factors regulating C. difficile pathogenicity are unknown. In recent years, it appeared that small non-coding RNAs (sRNAs) and RNA-binding proteins (RBPs) are involved in post-transcriptional regulation of nearly every facet of bacterial life. Most research has been conducted in Gram-negative species, generating a big knowledge gap in Gram-positives such as C. difficile. Despite ongoing efforts to unravel the functions of selected sRNAs and the RNA chaperone Hfq, we have only scratched the surface of studying post-transcriptional control in this pathogen. In this study, the complexomic approach gradient profiling by sequencing (Grad-seq) was applied to draft the landscape of cellular RNA and protein complexes in C. difficile. The sedimentation profiles of 3,541 transcripts (~88% of annotated transcripts) and 1,867 proteins (~50% of annotated proteins) were captured. Comparative analysis of the profiles reproduced major ribonucleoprotein particles (RNPs) and protein-protein interactions (PPIs). Based on several examples, the potential use of Grad-seq to imply protein function and discover unknown regulatory RNAs was demonstrated. With an established pull-down approach, the proteins KhpA and KhpB were identified as common interaction partners of similar behaving sRNAs. By applying crosslinking immunoprecipitation followed by RNA-sequencing (CLIP seq), a pervasive targetome of KhpB was revealed, comprising over one-third of all annotated transcripts, including sRNAs, tRNAs and untranslated regions. The scope of RNA-binding suggests that KhpB is a second globally acting RBP in C. difficile, next to Hfq. The combination of transcriptome studies and phenotypic characterization suggests that KhpA and KhpB have several physiological functions including the regulation of toxins and metabolic pathways associated with pathogenesis. Overall, this work provides a comprehensive dataset for the discovery of PPIs and RNA-protein interactions in C. difficile. Further, this study establishes the foundation for studying two novel players in post-transcriptional regulation in this important human pathogen.

Accumulation of lipid droplets (LD) and ceramides (Cer) is associated with the pathogenesis of metabolic dysfunction-associated steatotic liver disease (MASLD) a condition frequently co-occurring with type 2 diabetes and diminished immune functionality. Chronic inflammation and increased disease severity during viral infections characterize obesity-associated immunopathology. Upregulation of neutral sphingomyelinase-2 (NSM2) expression has been implicated in obesity-associated tissue pathology. NSM2 is a neutral sphingomyelinase associated with the inner leaflet of the plasma membrane (PM) where it hydrolyzes sphingomyelin into Cer and phosphocholine. The enzymatic activity of NSM2 therefore affects Cer and its associated protein content and the organization within the distinct PM nanodomains. Furthermore, NSM2 is crucial in the maintenance of cholesterol homeostasis and mitochondrial energy metabolism in T cells, indicating the crosstalk between NSM2-regulated sphingolipids and general lipid homeostasis. However, the molecular mechanisms of the NSM2 communication with sterol and lipid biosynthetic pathways are poorly understood. Especially, the role of sphingolipids, and particularly of NSM2, in modulating the immune cell responses in environments rich in fatty acids (FA) remains unexplored. In parallel to this doctoral study, the lipid droplet residing protein perilipin 3 (PLIN3) was identified within the intracellular nano-environment of NSM2 in Jurkat cells by employing the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. Super-resolution structured illumination microscopy (SIM) performed in this study confirmed the co-localization of NSM2 and PLIN3 within LD organelles which were induced by elevated extracellular concentrations of oleic acid (OA) in Jurkat cells. Moreover, the lipid analysis of the isolated LDs containing enzymatically active NSM2 revealed augmented Cer levels in these lipid storage organelles. Notably, NSM2 enzymatic activity was dispensable for its association with LDs but exerted a negative impact on LD numbers and overall cellular content of the neutral lipids triacylglycerols (TAGs) which are stored within LDs. The most negatively affected was the long-chain unsaturated TAG species. These findings correlated with the significantly reduced activity of enzymes producing diacylglycerol (DAG) and phosphatidic acid (PA) within the TAG biosynthesis pathway in Jurkat cells overexpressing enzymatically active NSM2. Concurrently, NSM2 activity stimulated mitochondrial respiration and FA β-oxidation (FAO) in response to increased OA levels, thereby inducing a shift toward a highly energetic state of the cells. The pharmacological inhibition of endogenous NSM2 activity in primary human CD4+ T cells confirmed the regulatory role of NSM2 in LD accumulation of T cell receptor (TCR) stimulated cells in FA-rich environment. Importantly, NSM2 activity was crucial for the proliferation and survival of activated T cells at high levels of OA in cell culture. Taken together, this study shows a novel intracellular localization of NSM2 within LDs and underscores the role of enzymatically active NSM2 and Cer in lipid storage organelles in shifting the cellular lipid metabolism from storage towards energy producing mitochondrial FAO thereby supporting T cell survival and proliferation in OA-rich environment.

Background Serotonin is a phylogenetically ancient molecule that is widely distributed in most metazoans, including flatworms. In addition to its role as a neurotransmitter, serotonin acts as a morphogen and regulates developmental processes. Although several studies have focused on the serotonergic nervous system in parasitic flatworms, little is known on the role of serotonin in flatworm development. Methods To study the effects of serotonin on proliferation and development of the cestode Echinococcus multilocularis, we cloned the genes encoding the E. multilocularis serotonin transporter (SERT) and tryptophan hydroxylase (TPH), analyzed gene expression by transcriptome analysis and whole mount in situ hybridization (WMISH) and performed cell culture experiments. Results We first characterized orthologues encoding the SERT and TPH, the rate-limiting enzyme in serotonin biosynthesis. WMISH and transcriptomic analyses indicated that the genes for both SERT and TPH are expressed in the parasite nervous system. Long-term treatment of parasite stem cell cultures with serotonin stimulated development towards the parasite metacestode stage. Mature metacestode vesicles treated with serotonin showed increased rates of incorporation of the thymidine analogue 5-ethynyl-2′-deoxyuridine (EdU), indicating stimulated cell proliferation. In contrast, treatment with the selective serotonin reuptake inhibitor paroxetine strongly affected the viability of parasite cells. Paroxetine also caused structural damage in metacestode vesicles, suggesting that serotonin transport is crucial for the integrity of parasite vesicles. Conclusions Our results indicate that serotonin plays an important role in E. multilocularis development and proliferation, providing evidence that the E. multilocularis SERT and TPH are expressed in the nervous system of the protoscolex. Our results further suggest that the E. multilocularis SERT has a secondary role outside the nervous system that is essential for parasite integrity and survival. Since serotonin stimulated E. multilocularis metacestode development and proliferation, serotonin might also contribute to the formation and growth of the parasite in the liver.

Allogeneic hematopoietic cell transplantation (allo-HCT) is increasingly used in older myelofibrosis (MF) patients, but its risk/benefit ratio compared to non-transplant approaches has not been evaluated in this population. We analyzed the outcomes of allo-HCT in 556 MF patients aged ≥65 years from the EBMT registry, and determined the excess mortality over the matched general population of MF patients ≥65 years managed with allo-HCT (n = 556) or conventional drug treatment (n = 176). The non-transplant cohort included patients with intermediate-2 or high risk DIPSS from the Spanish Myelofibrosis Registry. After a median follow-up of 3.4 years, the estimated 5-year survival rate, non-relapse mortality (NRM), and relapse incidence after transplantation was 40%, 37%, and 25%, respectively. Busulfan-based conditioning was associated with decreased mortality (HR: 0.7, 95% CI: 0.5–0.9) whereas the recipient CMV+/donor CMV- combination (HR: 1.7, 95% CI: 1.2–2.4) and the JAK2 mutated genotype (HR: 1.9, 95% CI: 1.1–3.5) predicted higher mortality. Busulfan-based conditioning correlated with improved survival due to less NRM, despite its higher relapse rate when compared with melphalan-based regimens. Excess mortality was higher in transplanted patients than in the non-HCT cohort in the first year of follow-up (ratio: 1.93, 95% CI: 1.13–2.80), whereas the opposite occurred between the fourth and eighth follow-up years (ratio: 0.31, 95% CI: 0.18–0.53). Comparing the excess mortality of the two treatments, male patients seemed to benefit more than females from allo-HCT, mainly due to their worse prognosis with non-transplant approaches. These findings could potentially enhance counseling and treatment decision-making in elderly transplant-eligible MF patients.