Characterization of Ceramide synthase 2 dysfunctions caused by targeted mutations in transgenic mice

Ceramide species are the precursor of sphingolipids, which serve as structural components of biological membranes or are bioactive molecules. In mammals, ceramides are synthesised by a family of ceramide synthases (CerS) 1 to 6. They catalyse the N-acylation of a sphingoid long chain base and a fatty acyl-CoA of varying chain length. All ceramide synthases harbour a lag1p motif essentially necessary for catalytic activity and all CerS, except CerS1, contain a homeodomain of unknown function. During this PhD thesis, different CerS2-overexpression constructs were generated and investigated to identify mutations suitable to analyse the catalytic function independent from homeodomain function of the mCerS2 protein. Consequently, two different CerS2 conditional mouse lines were generated, CerS2H212A/H213A (H/A) and CerS2Del79-120 (Del). Both mouse lines show a high mortality rate and develop severe phenotypic alterations. In H/A mice, the coding region of cers2 was exchanged by a CerS2H212A/H213A-eGFP fragment, resulting in the expression of a CerS2 point-mutated protein. Additional IRES-driven GPF-expression was used to monitor cers2H/A expression and to verify the presence of the CerS2H/A protein. In vitro assays confirmed that catalytic activity of CerS2 is lost in H/A mice. Additional qRT-PCR and immunoblot analyses confirmed expression of the CerS2H/A protein in all analysed tissues. Expression levels (mRNA or protein) however do not always correlate with that of WT control. Furthermore, the H/A replacement-mutation results in alterations of lipid and energy metabolism. First, Mass spectrometric analysis showed strong reduction in C22/ C24/ C24:1 ceramide species and an increase in long-chain ceramides (C16/C18), which does not restore the total sphingolipid content. Analysis regarding the storage lipid content of liver highlight significant reduction in TAG, DAG and FFA levels in mutated mice, which is accompanied by reduction in white adipose tissue (WAT) mass. Morphological and histological investigations of H/A mice revealed reduced body weight as well as size, while adipocyte size is even more disproportionately reduced. On standard diet, H/A mice ingest more food, but show less weight gain, which is not based on altered intestinal lipid absorption. Liver sections showed loss of lipid droplet formation and an unexpected loss of glycogen-content. Moreover, catalytic ablation also leads to the development of hepatocellular carcinoma. Transcriptome analyses performed in combination with detailed qRT-PCR analyses show down regulation of genes involved in lipid metabolism. Immunological analyses of H/A mice showed that depletion of catalytic activity and loss of corresponding ceramide species negatively affects egress of mature single positive T cells from the thymus into the periphery. Accumulation of naïve T cells within the thymus results in a mild T cell lymphopenia. The CerS2Del79-120 mutation was verified in cell culture experiments first. After transient transfection of the CerS2Del79-120 plasmid, the mutated CerS2 protein was expressed and further investigations indicate, that the deletion within the homeodomain had no effect on the catalytic activity. Yet, the Del mice show complete loss of the CerS2Del protein, leading to a loss of catalytic activity and a lack of C22-24 ceramide species. Del mice show the same phenotype as the CerS2gt/gt and H/A mice. All findings indicate that the catalytic domain of CerS2 is important for the synthesis of C22/C24 ceramide species. It is also necessary to maintain CerS2s regulatory functions in lipid and energy metabolism and to facilitate those functions, which allow the thymic egress of mature T cells

Systemic alterations in neutrophils and their precursors in early-stage chronic obstructive pulmonary disease

Summary: Systemic inflammation is established as part of late-stage severe lung disease, but molecular, functional, and phenotypic changes in peripheral immune cells in early disease stages remain ill defined. Chronic obstructive pulmonary disease (COPD) is a major respiratory disease characterized by small-airway inflammation, emphysema, and severe breathing difficulties. Using single-cell analyses we demonstrate that blood neutrophils are already increased in early-stage COPD, and changes in molecular and functional neutrophil states correlate with lung function decline. Assessing neutrophils and their bone marrow precursors in a murine cigarette smoke exposure model identified similar molecular changes in blood neutrophils and precursor populations that also occur in the blood and lung. Our study shows that systemic molecular alterations in neutrophils and their precursors are part of early-stage COPD, a finding to be further explored for potential therapeutic targets and biomarkers for early diagnosis and patient stratification

Disease severity-specific neutrophil signatures in blood transcriptomes stratify COVID-19 patients

Background!#!The SARS-CoV-2 pandemic is currently leading to increasing numbers of COVID-19 patients all over the world. Clinical presentations range from asymptomatic, mild respiratory tract infection, to severe cases with acute respiratory distress syndrome, respiratory failure, and death. Reports on a dysregulated immune system in the severe cases call for a better characterization and understanding of the changes in the immune system.!##!Methods!#!In order to dissect COVID-19-driven immune host responses, we performed RNA-seq of whole blood cell transcriptomes and granulocyte preparations from mild and severe COVID-19 patients and analyzed the data using a combination of conventional and data-driven co-expression analysis. Additionally, publicly available data was used to show the distinction from COVID-19 to other diseases. Reverse drug target prediction was used to identify known or novel drug candidates based on finding from data-driven findings.!##!Results!#!Here, we profiled whole blood transcriptomes of 39 COVID-19 patients and 10 control donors enabling a data-driven stratification based on molecular phenotype. Neutrophil activation-associated signatures were prominently enriched in severe patient groups, which was corroborated in whole blood transcriptomes from an independent second cohort of 30 as well as in granulocyte samples from a third cohort of 16 COVID-19 patients (44 samples). Comparison of COVID-19 blood transcriptomes with those of a collection of over 3100 samples derived from 12 different viral infections, inflammatory diseases, and independent control samples revealed highly specific transcriptome signatures for COVID-19. Further, stratified transcriptomes predicted patient subgroup-specific drug candidates targeting the dysregulated systemic immune response of the host.!##!Conclusions!#!Our study provides novel insights in the distinct molecular subgroups or phenotypes that are not simply explained by clinical parameters. We show that whole blood transcriptomes are extremely informative for COVID-19 since they capture granulocytes which are major drivers of disease severity