Institut für Virologie und Immunbiologie
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Modulation CD4+ humaner Treg- und Tconv-Zellen durch Inhibition der sauren Sphingomyelinase in vitro
(2020)
Die saure Sphingomyelinase (ASM) stellt durch die Umwandlung von Sphingomyelin in Ceramid und Phosphorylcholin ein zentrales, fein reguliertes Enzym im Sphingolipidmetabolismus dar. Dadurch nimmt es Einfluss auf verschiedene zelluläre Mechanismen wie Signalvermittlung, Endo- und Exozytose und Zellaktivierung. Dementsprechend weitreichend ist auch die Bedeutung der ASM bei verschiedenen Krankheiten wie Arteriosklerose, Depression oder Neoplasien. Auch auf das Immunsystem, insbesondere auf die Signalvermittlung durch T-Zellen innerhalb des adaptiven Immunsystems, nimmt die saure Sphingomyelinase Einfluss. Aufbauend auf früheren Forschungsarbeiten zur pharmakologischen und genetischen Hemmung der ASM im Mausmodell untersuchten wir, welche Auswirkungen die Hemmung dieses Enzyms in humanen Zellkulturen auf die Population regulatorischer und konventioneller T-Zellen haben. Hierzu verwendeten wir die beiden selektiven Serotonin-Wiederaufnahmehemmer Sertralin und Citalopram; zwei antidepressiv wirksame Medikamente, die durch eine Verdrängung der ASM von der lysosomalen Membran eine hemmende Wirkung ausüben. Wir konnten zeigen, dass diese beiden Substanzen sowohl in Maus-T-Zellen, als auch in humanen T-Zellen, in der Lage sind, die Aktivität der sauren Sphingomyelinase zu inhibieren. Durch Kultivierung von Immunzellen der Maus zusammen mit den Inhibitoren konnte darüber hinaus eine Erhöhung der Treg-Zellfrequenz erreicht werden. Verschiedene Zellkulturexperimente mit humanen PBMCs zeigten weiterhin, dass unter gewissen Umständen so auch eine Vermehrung regulatorischer T-Zellen im Menschen möglich ist, und dass dies mutmaßlich durch Einbindung der ASM im CD3/CD28-Signalweg bedingt ist. In mit AntiCD3-Antikörper stimulierten experimentellen Ansätzen kam es jedoch nur bei einzelnen Individuen, die als Responder identifiziert werden konnten, zu einer Treg-Zellvermehrung. Umgekehrt kam es durch externe Zugabe von C6-Ceramid zu einer Verringerung des Anteils an regulatorischen T-Zellen. Des Weiteren wurden verschiedene Veränderungen im Expressionsverhalten von Treg- und Tconv-Zellen bezüglich CD25, CD69 und CTLA-4 in Anwesenheit der ASMInhibitoren beobachtet. Weiterhin bestätigte sich, dass die pharmakologische Hemmung der sauren Sphingomyelinase auch Auswirkungen auf die Effektorfunktion von T-Zellen hat. Während die Proliferation der Zellen weitgehend unbeeinträchtigt blieb, kam es zu einer verringerten Sekretion der Zytokine IFN-gamma, TNF, IL-5 und IL-10. In ihrer Gesamtheit sprechen diese Ergebnisse dafür, dass Inhibitoren der sauren Sphingomyelinase begünstigend auf Krankheitsgeschehen mit überschießender oder dysregulierter Aktivität des Immunsystems einwirken könnten. Immunmodulatorischen Wirkungen durch Inhibition der ASM erklären möglicherweise auch Einflüsse auf das Immunsystem, die für verschiedene Antidepressiva beschrieben wurden. Insgesamt ist die Bedeutung der sauren Sphingomyelinase innerhalb der Regulation des adaptiven Immunsystems jedoch noch ein weitgehend ungeklärtes Thema mit vielen offenen Fragen. Daher ist auch in Zukunft weitere klinische und experimentelle Forschung erforderlich, um zu klären, welchen Einfluss dieses Enzyms auf Immunzellen hat und wie sich dieser auch klinisch anwenden lässt.
Donor CD4\(^+\)Foxp3\(^+\) regulatory T cells (T reg cells) suppress graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (HCT allo-HCT]). Current clinical study protocols rely on the ex vivo expansion of donor T reg cells and their infusion in high numbers. In this study, we present a novel strategy for inhibiting GvHD that is based on the in vivo expansion of recipient T reg cells before allo-HCT, exploiting the crucial role of tumor necrosis factor receptor 2 (TNFR2) in T reg cell biology. Expanding radiation-resistant host T reg cells in recipient mice using a mouse TNFR2-selective agonist before allo-HCT significantly prolonged survival and reduced GvHD severity in a TNFR2-and T reg cell-dependent manner. The beneficial effects of transplanted T cells against leukemia cells and infectious pathogens remained unaffected. A corresponding human TNFR2-specific agonist expanded human T reg cells in vitro. These observations indicate the potential of our strategy to protect allo-HCT patients from acute GvHD by expanding T reg cells via selective TNFR2 activation in vivo.
Tuberculosis patients and mice infected with live Mycobacterium tuberculosis accumulate high numbers of myeloid-derived suppressor cells (MDSCs). Here, we hypothesized that dead M. tuberculosis vaccines also may induce MDSCs that could impair the efficacy of vaccination. We found that repeated injections of M. tuberculosis vaccines (heat-killed M. tuberculosis in incomplete Freund’s adjuvant, such as Montanide) but not single or control vaccines without M. tuberculosis strongly expanded CD11b\(^+\) myeloid cells in the spleen, leading to T cell suppression of proliferation and killing ex vivo. Dead M. tuberculosis vaccination induced the generation of CD11b\(^+\)Ly6C\(^{hi}\)CD115\(^+\) iNOS/Nos2\(^+\) monocytic MDSCs (M-MDSCs) upon application of inflammatory or microbial activation signals. In vivo these M-MDSCs were positioned strategically in the splenic bridging channels and then positioned in the white pulp areas. Notably, within 6–24 hours, in a Nos2-dependent fashion, they produced NO to rapidly kill conventional and plasmacytoid DCs while, surprisingly, sparing T cells in vivo. Thus, we demonstrate that M. tuberculosis vaccine induced M-MDSCs do not directly suppress effector T cells in vivo but, instead, indirectly by killing DCs. Collectively, we demonstrate that M. tuberculosis booster vaccines induce M-MDSCs in the spleen that can be activated to kill DCs. Our data suggest that formation of MDSCs by M. tuberculosis vaccines should be investigated also in clinical trials.
Targeting bromodomain-containing protein 4 (BRD4) inhibits MYC expression in colorectal cancer cells
(2019)
The transcriptional regulator BRD4 has been shown to be important for the expression of several oncogenes including MYC. Inhibiting of BRD4 has broad antiproliferative activity in different cancer cell types. The small molecule JQ1 blocks the interaction of BRD4 with acetylated histones leading to transcriptional modulation. Depleting BRD4 via engineered bifunctional small molecules named PROTACs (proteolysis targeting chimeras) represents the next-generation approach to JQ1-mediated BRD4 inhibition. PROTACs trigger BRD4 for proteasomale degradation by recruiting E3 ligases. The aim of this study was therefore to validate the importance of BRD4 as a relevant target in colorectal cancer (CRC) cells and to compare the efficacy of BRD4 inhibition with BRD4 degradation on downregulating MYC expression. JQ1 induced a downregulation of both MYC mRNA and MYC protein associated with an antiproliferative phenotype in CRC cells. dBET1 and MZ1 induced degradation of BRD4 followed by a reduction in MYC expression and CRC cell proliferation. In SW480 cells, where dBET1 failed, we found significantly lower levels of the E3 ligase cereblon, which is essential for dBET1-induced BRD4 degradation. To gain mechanistic insight into the unresponsiveness to dBET1, we generated dBET1-resistant LS174t cells and found a strong downregulation of cereblon protein. These findings suggest that inhibition of BRD4 by JQ1 and degradation of BRD4 by dBET1 and MZ1 are powerful tools for reducing MYC expression and CRC cell proliferation. In addition, downregulation of cereblon may be an important mechanism for developing dBET1 resistance, which can be evaded by incubating dBET1-resistant cells with JQ1 or MZ1.
Spread and clinical severity of respiratory syncytial virus A genotype ON1 in Germany, 2011–2017
(2019)
Background
The Respiratory Syncytial Virus (RSV) A genotype ON1, which was first detected in Ontario (Canada) in 2010/11, appeared in Germany in 2011/12. Preliminary observations suggested a higher clinical severity in children infected with this new genotype. We investigated spread and disease severity of RSV-A ON1 in pediatric in- and outpatient settings.
Methods
During 2010/11 to 2016/17, clinical characteristics and respiratory samples from children with acute respiratory tract infections (RTI) were obtained from ongoing surveillance studies in 33 pediatric practices (PP), one pediatric hospital ward (PW) and 23 pediatric intensive care units (PICU) in Germany. RSV was detected in the respiratory samples by PCR; genotypes were identified by sequencing. Within each setting, clinical severity markers were compared between RSV-A ON1 and RSV-A non-ON1 genotypes.
Results
A total of 603 children with RSV-RTI were included (132 children in PP, 288 in PW, and 183 in PICU). Of these children, 341 (56.6%) were infected with RSV-A, 235 (39.0%) with RSV-B, and one child (0.2%) with both RSV-A and RSV-B; in 26 (4.3%) children, the subtype could not be identified. In the 341 RSV-A positive samples, genotype ON1 was detected in 247 (72.4%), NA1 in 92 (26.9%), and GA5 in 2 children (0.6%). RSV-A ON1, rarely observed in 2011/12, was the predominant RSV-A genotype in all settings by 2012/13 and remained predominant until 2016/17. Children in PP or PW infected with RSV-A ON1 did not show a more severe clinical course of disease compared with RSV-A non-ON1 infections. In the PICU group, hospital stay was one day longer (median 8 days, inter-quartile range (IQR) 7–12 vs. 7 days, IQR 5–9; p = 0.02) and duration of oxygen treatment two days longer (median 6 days, IQR 4–9 vs. 4 days, IQR 2–6; p = 0.03) for children infected with RSV-A ON1.
Conclusions
In children, RSV-A ON1 largely replaced RSV-A non-ON1 genotypes within two seasons and remained the predominant RSV-A genotype in Germany during subsequent seasons. A higher clinical severity of RSV-A ON1 was observed within the group of children receiving PICU treatment, whereas in other settings clinical severity of RSV-A ON1 and non-ON1 genotypes was largely similar.
Transmission of measles virus (MV) from dendritic to airway epithelial cells is considered as crucial to viral spread late in infection. Therefore, pathways and effectors governing this process are promising targets for intervention. To identify these, we established a 3D respiratory tract model where MV transmission by infected dendritic cells (DCs) relied on the presence of nectin-4 on H358 lung epithelial cells. Access to recipient cells is an important prerequisite for transmission, and we therefore analyzed migration of MV-exposed DC cultures within the model. Surprisingly, enhanced motility toward the epithelial layer was observed for MV-infected DCs as compared to their uninfected siblings. This occurred independently of factors released from H358 cells indicating that MV infection triggered cytoskeletal remodeling associated with DC polarization enforced velocity. Accordingly, the latter was also observed for MV-infected DCs in collagen matrices and was particularly sensitive to ROCK inhibition indicating infected DCs preferentially employed the amoeboid migration mode. This was also implicated by loss of podosomes and reduced filopodial activity both of which were retained in MV-exposed uninfected DCs. Evidently, sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) as produced in response to virus-infection in DCs contributed to enhanced velocity because this was abrogated upon inhibition of sphingosine kinase activity. These findings indicate that MV infection promotes a push-and-squeeze fast amoeboid migration mode via the SphK/S1P system characterized by loss of filopodia and podosome dissolution. Consequently, this enables rapid trafficking of virus toward epithelial cells during viral exit.
Enterovirus D68 (EV-D68) has been recognised as a worldwide emerging pathogen associated with severe respiratory symptoms since 2009. We here report EV-D68 detection in hospitalised patients with acute respiratory infection admitted to three tertiary hospitals in Germany between January 2013 and December 2014. From a total of 14,838 respiratory samples obtained during the study period, 246 (1.7%) tested enterovirus-positive and, among these, 39 (15.9%) were identified as EV-D68. Infection was observed in children and teenagers (0–19 years; n=31), the majority (n=22) being under five years-old, as well as in adults > 50 years of age (n=8). No significant difference in prevalence was observed between the 2013 and 2014 seasons. Phylogenetic analyses based on viral protein 1 (VP1) sequences showed co-circulation of different EV-D68 lineages in Germany. Sequence data encompassing the entire capsid region of the genome were analysed to gain information on amino acid changes possibly relevant for immunogenicity and revealed mutations in two recently described pleconaril binding sites.
Genome-wide association studies revealed CLEC16A as a candidate gene for Type 1 Diabetes and multiple other autoimmune disorders. The function of CLEC16A remains unknown. However, previous work showed that the CLEC16A ortholog ema and the murine Clec16a were both implicated in autophagy, a process partially required for MHC class II loading and antigen presentation. Furthermore, studies could show that autophagy was required in thymic epithelial cells for antigen presentation during T cell selection, suggesting a possible role of CLEC16A in T cell selection in the thymus. Additionally, it was postulated that CLEC16A may function as an expression quantitative trait locus for its neighboring genes and that Clec16a KD was involved in pancreatic islet function and impaired insulin secretion and glucose homeostasis. Prior to this work, Schuster et al. had created a Clec16a KD NOD mouse, which was protected from spontaneous autoimmune diabetes.
For this work it was hypothesized that CLEC16A variation serves as a Type 1 Diabetes risk gene by affecting autophagy in thymic epithelial cells, which modulates antigen presentation and shapes the T cell repertoire. To expand and complement previous findings by Schuster et al., this thesis aimed to investigate how CLEC16A modifies the function of thymic epithelial cells. For this purpose, CLEC16A KD was induced in human cells via RNA interference and autophagy was studied through immunoblotting. Additionally, inflammation of pancreatic tissue in Clec16a KD NOD mice was scored using H.E. stained pancreatic sections. Thymic transplantation experiments were conducted to test whether the effects of Clec16a KD were T cell intrinsic. Also, intraperitoneal glucose tolerance tests were performed to study glucose homeostasis in Clec16a KD NOD animals. Finally, using qPCR, gene expression levels of neighboring genes such as Dexi and Socs1 were measured to study Clec16a as an expression quantitative trait locus.
In combination with the findings of Schuster et al., this thesis demonstrates that Clec16a KD reduces the severity of insulitis and protects from onset of spontaneous diabetes in the NOD mouse. Disease protection is conveyed by impaired autophagy in TEC, which leads to altered T cell selection and hyporeactive CD4+ T cells. The effects of Clec16a KD in the NOD mouse are thymus intrinsic. Glucose homeostasis remains unchanged in the Clec16a KD NOD mouse and plays no role in disease protection. Clec16a and Dexi presented similar expression levels, but further studies are required to investigate a clear link between these two genes. Finally, impaired autophagy could be replicated in human CLEC16A KD cells, which demonstrates a conserved function of CLEC16A and suggests a possible link between CLEC16A variation and risk of autoimmune disease in human.
In T cells, as in all other cells of the body, sphingolipids form important structural components of membranes. Due to metabolic modifications, sphingolipids additionally play an active part in the signaling of cell surface receptors of T cells like the T cell receptor or the co-stimulatory molecule CD28. Moreover, the sphingolipid composition of their membranes crucially affects the integrity and function of subcellular compartments such as the lysosome. Previously, studying sphingolipid metabolism has been severely hampered by the limited number of analytical methods/model systems available. Besides well-established high resolution mass spectrometry new tools are now available like novel minimally modified sphingolipid subspecies for click chemistry as well as recently generated mouse mutants with deficiencies/overexpression of sphingolipid-modifying enzymes. Making use of these tools we and others discovered that the sphingolipid sphingomyelin is metabolized to ceramide to different degrees in distinct T cell subpopulations of mice and humans. This knowledge has already been translated into novel immunomodulatory approaches in mice and will in the future hopefully also be applicable to humans. In this paper we are, thus, summarizing the most recent findings on the impact of sphingolipid metabolism on T cell activation, differentiation, and effector functions. Moreover, we are discussing the therapeutic concepts arising from these insights and drugs or drug candidates which are already in clinical use or could be developed for clinical use in patients with diseases as distant as major depression and chronic viral infection.
As structural membrane components and signaling effector molecules sphingolipids influence a plethora of host cell functions, and by doing so also the replication of viruses. Investigating the effects of various inhibitors of sphingolipid metabolism in primary human peripheral blood lymphocytes (PBL) and the human B cell line BJAB we found that not only the sphingosine kinase (SphK) inhibitor SKI-II, but also the acid ceramidase inhibitor ceranib-2 efficiently inhibited measles virus (MV) replication. Virus uptake into the target cells was not grossly altered by the two inhibitors, while titers of newly synthesized MV were reduced by approximately 1 log (90%) in PBL and 70–80% in BJAB cells. Lipidomic analyses revealed that in PBL SKI-II led to increased ceramide levels, whereas in BJAB cells ceranib-2 increased ceramides. SKI-II treatment decreased sphingosine-1-phosphate (S1P) levels in PBL and BJAB cells. Furthermore, we found that MV infection of lymphocytes induced a transient (0.5–6 h) increase in S1P, which was prevented by SKI-II. Investigating the effect of the inhibitors on the metabolic (mTORC1) activity we found that ceranib-2 reduced the phosphorylation of p70 S6K in PBL, and that both inhibitors, ceranib-2 and SKI-II, reduced the phosphorylation of p70 S6K in BJAB cells. As mTORC1 activity is required for efficient MV replication, this effect of the inhibitors is one possible antiviral mechanism. In addition, reduced intracellular S1P levels affect a number of signaling pathways and functions including Hsp90 activity, which was reported to be required for MV replication. Accordingly, we found that pharmacological inhibition of Hsp90 with the inhibitor 17-AAG strongly impaired MV replication in primary PBL. Thus, our data suggest that treatment of lymphocytes with both, acid ceramidase and SphK inhibitors, impair MV replication by affecting a number of cellular activities including mTORC1 and Hsp90, which alter the metabolic state of the cells causing a hostile environment for the virus.
