Die Spezies-spezifische Rolle der Caspase-8 als Regulator der Nekroptose

The molecular mechanisms of the necroptotic signaling pathway remain to a certain proportion unknown. Since necroptosis is associated to various diseases, a precise knowledge of the necroptotic signaling pathway is essential for the development of new therapeutic strategies. The aim of this dissertation was to therefore gain a deeper insight into molecular events of TNF-induced necroptosis. In the first part of the thesis, murine L929Ts cells deficient for caspase-8 as well as human HT-29 cells deficient for caspase-8 or caspase-10 were generated by employing the CRISPR/Cas9 technology. It was shown that genetic ablation of caspase-8 in murine L929Ts cells enhances necroptosis, whereas genetic ablation of caspase-8 in human HT-29 cells protects the cells from TNF-induced necroptosis. It is noteworthy that humans express caspase-8 and caspase-10, while caspase-10 was lost in mice over the course of evolution. In the presented dissertation, it was demonstrated that genetic ablation of caspase-10 in human cells exacerbates necroptosis, supporting the hypothesis that caspase-10 in humans can substitute the function that is held by caspase-8 in mice. Furthermore, experiments aimed at the identification of differences in the composition of necrosomes in wild type vs. caspase-8- or caspase-10-deficient HT-29 cells found that caspase-10 associates with caspase-8 during TNF-induced necroptosis. Genetic ablation of caspase-8 disrupts the assembly of the necrosome while genetic ablation of caspase-10 promotes at least the recruitment of RIPK1 and RIPK3. Additionally, data obtained in this thesis revealed that the enzymatic activity of human and mouse caspase-8, as well as the enzymatic activity of caspase-10, are required for their function within the necroptotic signaling pathway. In summary, it can be stated that all data obtained in this thesis consistently point to a differential proteolytic regulation of necroptosis by caspase-8 in mice and in humans.Da Nekroptose mit vielen verschiedenen Krankheiten in Verbindung steht, ist es essentiell, genaue Erkenntnis über den Signalweg der Nekroptose zu erlangen, um neue therapeutische Strategien entwickeln zu können. Daher war das Ziel dieser Dissertation, einen tieferen Einblick in molekulare Prozesse der TNF-induzierten Nekroptose zu gewinnen. Im ersten Teil dieser Arbeit wurden sowohl Caspase-8-defiziente L929Ts Mauszellen als auch Caspase-8- oder Caspase-10-defiziente humane HT-29 Zellen mittels CRISPR/Cas9 Technologie generiert. Es wurde gezeigt, dass genetische Deletion von Caspase-8 in L929Ts Mauszellen Nekroptose verstärkt, wohingegen genetische Deletion von Caspase-8 in humanen HT-29 Zellen die Zellen vor TNF-induzierter Nekroptose schützt. Nennenswert an dieser Stelle ist, dass der Mensch Caspase-8 und Caspase-10 exprimiert, während Caspase-10 in Mäusen im Verlauf der Evolution verloren gegangen ist. In der vorliegenden Dissertation wurde gezeigt, dass genetische Deletion von Caspase-10 in humanen Zellen Nekroptose begünstigt, was die Hypothese unterstützt, dass Caspase-10 im Menschen die Funktion, welche der Caspase-8 in Mäusen zugeschrieben ist, übernimmt. Des Weiteren haben Experimente mit dem Ziel zur Identifikation von Unterschieden in der Komposition des „Nekrosoms“ von Wildtyp vs. Caspase-8-defizienten HT-29 Zellen ergeben, dass Caspase-10 und Caspase-8 während TNF-induzierter Nekroptose assoziieren und dass die genetische Deletion von Caspase-8 die Rekrutierung von Caspase-10 verhindert. Die genetische Deletion von Caspase-8 stört die Bildung des „Nekrosoms“, während die genetische Deletion von Caspase-10 zumindest die Rekrutierung von RIPK1 und RIPK3 begünstigt. Zusammenfassend kann gesagt werden, dass alle Daten, welche innerhalb der vorliegenden Arbeit generiert wurden, übereinstimmend auf einen Unterschied in der proteolytischen Regulation der Nekroptose durch Caspase-8 in der Maus und im Menschen hindeuten

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer‐reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state‐of‐the‐art handbook for basic and clinical researchers.DFG, 389687267, Kompartimentalisierung, Aufrechterhaltung und Reaktivierung humaner Gedächtnis-T-Lymphozyten aus Knochenmark und peripherem BlutDFG, 80750187, SFB 841: Leberentzündungen: Infektion, Immunregulation und KonsequenzenEC/H2020/800924/EU/International Cancer Research Fellowships - 2/iCARE-2DFG, 252623821, Die Rolle von follikulären T-Helferzellen in T-Helferzell-Differenzierung, Funktion und PlastizitätDFG, 390873048, EXC 2151: ImmunoSensation2 - the immune sensory syste

Differences and Similarities in TRAIL- and Tumor Necrosis Factor-Mediated Necroptotic Signaling in Cancer Cells.

Recently, a type of regulated necrosis (RN) called necroptosis was identified to be involved in many pathophysiological processes and emerged as an alternative method to eliminate cancer cells. However, only a few studies have elucidated components of TRAIL-mediated necroptosis useful for anticancer therapy. Therefore, we have compared this type of cell death to tumor necrosis factor (TNF)-mediated necroptosis and found similar signaling through acid and neutral sphingomyelinases, the mitochondrial serine protease HtrA2/Omi, Atg5, and vacuolar H(+)-ATPase. Notably, executive mechanisms of both TRAIL- and TNF-mediated necroptosis are independent of poly(ADP-ribose) polymerase 1 (PARP-1), and depletion of p38α increases the levels of both types of cell death. Moreover, we found differences in signaling between TNF- and TRAIL-mediated necroptosis, e.g., a lack of involvement of ubiquitin carboxyl hydrolase L1 (UCH-L1) and Atg16L1 in executive mechanisms of TRAIL-mediated necroptosis. Furthermore, we discovered indications of an altered involvement of mitochondrial components, since overexpression of the mitochondrial protein Bcl-2 protected Jurkat cells from TRAIL- and TNF-mediated necroptosis, and overexpression of Bcl-XL diminished only TRAIL-induced necroptosis in Colo357 cells. Furthermore, TRAIL does not require receptor internalization and endosome-lysosome acidification to mediate necroptosis. Taken together, pathways described for TRAIL-mediated necroptosis and differences from those for TNF-mediated necroptosis might be unique targets to increase or modify necroptotic signaling and eliminate tumor cells more specifically in future anticancer approaches

B Lymphocyte Stimulator (BLyS) Is Expressed in Human Adipocytes In Vivo and Is Related to Obesity but Not to Insulin Resistance

Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001). Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001) and are positively correlated to the BMI (r = 0.43, p<0.0002). In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d) had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance

Low-Avidity CD4(+) T Cell Responses to SARS-CoV-2 in Unexposed Individuals and Humans with Severe COVID-19

CD4 (+) T cells reactive against SARS-CoV-2 can be found in unexposed individuals, and these are suggested to arise in response to common cold coronavirus (CCCoV) infection. Here, we utilized SARS-CoV-2-reactive CD4(+) T cell enrichment to examine the antigen avidity and clonality of these cells, as well as the relative contribution of CCCoV cross-reactivity. SARS-CoV-2-reactive CD4(+) memory T cells were present in virtually all unexposed individuals examined, displaying low functional avidity and multiple, highly variable cross-reactivities that were not restricted to CCCoVs. SARS-CoV-2-reactive CD4 (+) T cells from COVID-1 9 patients lacked cross-reactivity to CCCoVs, irrespective of strong memory T cell responses against CCCoV in all donors analyzed. In severe but not mild COVID-19, SARS-CoV-2-specific T cells displayed low functional avidity and clonality, despite increased frequencies. Our findings identify low-avidity CD4(+) T cell responses as a hallmark of severe COVID-19 and argue against a protective role for CCCoV-reactive T cells in SARS-CoV-2 infection

The pre-exposure SARS-CoV-2-specific T cell repertoire determines the quality of the immune response to vaccination

SARS-CoV-2 infection and vaccination generates enormous host-response heterogeneity and an age -dependent loss of immune-response quality. How the pre-exposure T cell repertoire contributes to this heterogeneity is poorly understood. We combined analysis of SARS-CoV-2-specific CD4+ T cells pre-and post-vaccination with longitudinal T cell receptor tracking. We identified strong pre-exposure T cell variability that correlated with subsequent immune-response quality and age. High-quality responses, defined by strong expansion of high-avidity spike-specific T cells, high interleukin-21 production, and specific immuno-globulin G, depended on an intact naive repertoire and exclusion of pre-existing memory T cells. In the elderly, T cell expansion from both compartments was severely compromised. Our results reveal that an intrinsic defect of the CD4+ T cell repertoire causes the age-dependent decline of immune-response quality against SARS-CoV-2 and highlight the need for alternative strategies to induce high-quality T cell responses against newly arising pathogens in the elderly

BLyS serum levels in human obesity.

<p>(A) represents significant differences between different grades of obesity (grade 1+2 (BMI≥30 to <40 kg/m²), n = 20 and grade 3 (BMI≥40 kg/m²), n = 42) and normal-weight ((BMI≤25 kg/m²), n = 10) human control subjects. (B) shows the correlation (<i>r</i> = 0.43, p<0.0002) between BLyS serum levels and BMI of n = 72 human subjects used in this study. Data in this figure are shown as means+SD.</p

Baseline characteristics of the study populations of the BLyS mRNA in human total adipose tissue biopsies of lean, grade 3 obese +/− insulin resistance (IR) and of BLyS measurements in sera of lean, grade 1+2 obese +/− insulin resistance.

<p>t₁: lean vs. obese.</p><p>t₂: lean vs. obese+IR.</p><p>t₃: obese vs. obese+IR.</p><p>Shown are means+SD for each group.</p

BLyS mRNA level in (A) visceral and (B) subcutaneous adipose tissue of lean control subjects, patients with grade 3 obesity +/− type 2 diabetes (T2D).

<p>RNA was isolated from total subcutaneous and visceral adipose tissue biopsies. Data in this figure are shown as means+SD. (C) shows a significant negative correlation between age and BLyS expression (<i>r</i> = −0.48, p<0.0001) in a combined analysis of all biopsy samples.</p