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Since the advent of high-throughput sequencing technologies in the mid-2010s, RNA se-
quencing (RNA-seq) has been established as the method of choice for studying gene
expression. In comparison to microarray-based methods, which have mainly been used to
study gene expression before the rise of RNA-seq, RNA-seq is able to profile the entire
transcriptome of an organism without the need to predefine genes of interest. Today,
a wide variety of RNA-seq methods and protocols exist, including dual RNA sequenc-
ing (dual RNA-seq) and multi RNA sequencing (multi RNA-seq). Dual RNA-seq and
multi RNA-seq simultaneously investigate the transcriptomes of two or more species, re-
spectively. Therefore, the total RNA of all interacting species is sequenced together and
only separated in silico. Compared to conventional RNA-seq, which can only investi-
gate one species at a time, dual RNA-seq and multi RNA-seq analyses can connect the
transcriptome changes of the species being investigated and thus give a clearer picture of
the interspecies interactions. Dual RNA-seq and multi RNA-seq have been applied to a
variety of host-pathogen, mutualistic and commensal interaction systems.
We applied dual RNA-seq to a host-pathogen system of human mast cells and Staphylo-
coccus aureus (S. aureus). S. aureus, a commensal gram-positive bacterium, can become
an opportunistic pathogen and infect skin lesions of atopic dermatitis (AD) patients.
Among the first immune cells S. aureus encounters are mast cells, which have previously
been shown to be able to kill the bacteria by discharging antimicrobial products and re-
leasing extracellular traps made of protein and deoxyribonucleic acid (DNA). However,
S. aureus is known to evade the host’s immune response by internalizing within mast
cells. Our dual RNA-seq analysis of different infection settings revealed that mast cells
and S. aureus need physical contact to influence each other’s gene expression. We could
show that S. aureus cells internalizing within mast cells undergo profound transcriptome
changes to adjust their metabolism to survive in the intracellular niche. On the host side,
we found out that infected mast cells elicit a type-I interferon (IFN-I) response in an
autocrine manner and in a paracrine manner to non-infected bystander-cells. Our study
provides the first evidence that mast cells are capable to produce IFN-I upon infection
with a bacterial pathogen.
Additive manufacturing processes such as 3D printing are booming in the industry due to their high degree of freedom in terms of geometric shapes and available materials. Focusing on patient-specific medicine, 3D printing has also proven useful in the Life Sciences, where it exploits the shape fidelity for individualized tissues in the field of bioprinting. In parallel, the current systems of bioreactor technology have adapted to the new manufacturing technology as well and 3D-printed bioreactors are increasingly being developed. For the first time, this work combines the manufacturing of the tissue and a tailored bioreactor, significantly streamlining the overall process and optimally merging the two processes. This way the production of the tissues can be individualized by customizing the reactor to the tissue and the patient-specific wound geometry. For this reason, a common basis and guideline for the cross-device and cross-material use of 3D printers was created initially. Their applicability was demonstrated by the iterative development of a perfusable bioreactor system, made from polydimethylsiloxane (PDMS) and a lignin-based filament, into which a biological tissue of flexible shape can be bioprinted. Cost-effective bioink-replacements and in silico computational fluid dynamics simulations were used for material sustainability and shape development. Also, nutrient distribution and shear stress could be predicted in this way pre-experimentally.
As a proof of functionality and adaptability of the reactor, tissues made from a nanocellulose-based Cellink® Bioink, as well as an alginate-based ink mixed with Me-PMeOx100-b-PnPrOzi100-EIP (POx) (Alginate-POx bioink) were successfully cultured dynamically in the bioreactor together with C2C12 cell line. Tissue maturation was further demonstrated using hMSC which were successfully induced to adipocyte differentiation. For further standardization, a mobile electrical device for automated media exchange was developed, improving handling in the laboratory and thus reduces the probability of contamination.
The platelet cytoskeleton ensures normal size and discoid shape under resting conditions and undergoes immediate reorganization in response to changes in the extracellular environment through integrin-based adhesion sites, resulting in actomyosin-mediated contractile forces. Mutations in the contractile protein non-muscle myosin heavy chain IIA display, among others, macrothrombocytopenia and a mild to moderate bleeding tendency in human patients. It is insufficiently understood which factors contribute to the hemostatic defect found in MYH9-related disease patients. Therefore, a better understanding of the underlying biophysical mechanisms in thrombus formation and stabilization is warranted.
This thesis demonstrates that an amino acid exchange at the positions 702, 1424 and 1841 in the heavy chain of the contractile protein non-muscle myosin IIA, caused by heterozygous point mutations in the gene, resulted in macrothrombocytopenia and increased bleeding in mice, reflecting the clinical hallmark of the MYH9-related disease in human patients. Basic characterization of biological functions of Myh9 mutant platelets revealed overall normal surface glycoprotein expression and agonist-induced activation when compared to wildtype platelets. However, myosin light chain phosphorylation after thrombin-activation was reduced in mutant platelets, resulting in less contractile forces and a defect in clot retraction. Altered biophysical characteristics with lower adhesion and interaction forces of Myh9 mutant platelets led to reduced thrombus formation and stability. Platelets from patients with the respective mutations recapitulated the findings obtained with murine platelets, such as impaired thrombus formation and stiffness.
Besides biological and biophysical characterization of mutant platelets from mice and men, treatment options were investigated to prevent increased bleeding caused by reduced platelet forces. The antifibrinolytic agent tranexamic acid was applied to stabilize less compact thrombi, which are presumably more vulnerable to fibrinolysis. The hemostatic function in Myh9 mutant mice was improved by interfering with the fibrinolytic system. These results show the beneficial effect of fibrin stabilization to reduce bleeding in MYH9-related disease.
Studies on the role of cytoskeletal-regulatory and -crosslinking proteins in platelet function
(2023)
Cytoskeletal reorganization in platelets is highly regulated and important for proper platelet function during activation and aggregation at sites of vascular injury. In this thesis, the role of three different cytoskeletal-regulatory and -crosslinking proteins was studied in platelet physiology using megakaryocyte- and platelet-specific knockout mice. The generation of branched actin filaments is regulated by nucleation promoting factors (NPF) and the Arp2/3 complex.
(1.) The WAVE complex is a NPF, which upregulates the Arp2/3 complex activity at the plasma membrane. As shown in this thesis, the loss of the WAVE complex subunit Cyfip1 in mice did not alter platelet production and had only a minor impact on platelet activation. However, Cyfip1 played an essential role for branching of actin filaments and consequently for lamellipodia formation in vitro. The importance of lamellipodia for thrombus formation and stability has been controversially discussed. Cyfip1-deficient platelets were able to form a stable thrombus ex vivo and in vivo and a hemostatic plug comparable to controls. Moreover, Cyfip1-deficient mice maintained vascular integrity at the site of inflammation. These data show that platelet lamellipodia formation is not required for hemostatic function and pathophysiological thrombus formation.
(2.) The WASH complex is another NPF, which mediates actin filament polymerization on endosomal vesicles via the Arp2/3 complex. Loss of the WASH complex subunit Strumpellin led to a decreased protein abundance of the WASH protein and to a 20% reduction in integrin αIIbβ3 surface expression on platelets and megakaryocytes, whereas the expression of other surface receptors as well as the platelet count, size, ex vivo thrombus formation and bleeding time remained unaltered. These data point to a distinct role of Strumpellin in maintaining integrin αIIbβ3 expression and provide new insights into regulatory mechanisms of platelet integrins.
(3.) MACF1 has been described as a cytoskeletal crosslinker of microtubules and F-actin. However, MACF1-deficient mice displayed no alterations in platelet production, activation, thrombus formation and hemostatic function. Further, no compensatory up- or downregulation of other proteins could be found that contain an F-actin- and a microtubule-binding domain. These data indicate that MACF1 is dispensable for platelet biogenesis, activation and thrombus formation. Nevertheless, functional redundancy among different proteins mediating the cytoskeletal crosstalk may exist.
Ein Schlüsselereignis, welches dem prognosebestimmenden Organversagen bei systemi-schen Entzündungsprozessen und Sepsis vorangeht, ist die Entwicklung einer mikrovas-kulären endothelialen Schrankenstörung. Das vaskuläre endotheliale (VE-) Cadherin als mechanischer Stabilisator der Endothelbarriere spielt dabei eine wichtige Rolle. In der Inflammation werden Spaltprodukte von VE-Cadherin (sVE-Cadherin) gebildet. Ge-genstand der vorliegenden Arbeit war die Untersuchung der Hypothese ob diese Spalt-produkte selbst an der Störung der endothelialen Barrierefunktion beteiligt sind.
Es wurde hierfür humanes sVE-Cadherin bestehend aus den extrazellulären Domänen EC1-5 (sVE-CadherinEC1-5) generiert. In Messungen des transendothelialen elektrischen Widerstands (TER), mit Immunfluoreszenzfärbungen und Western Blot Analysen wird gezeigt, dass sVE-Cadherin dosisabhängig die Barriere Integrität in primären humanen dermalen Endothelzellen stört. Dies führt zu einer Reduktion von VE-Cadherin und den assoziierten Proteinen α-, γ- und δ-Catenin und ZO-1, die nach der Applikation von sVE-Cadherin an den Zellgrenzen reduziert sind. Die Interaktion zwischen VE-PTP und VE-Cadherin wird durch sVE-CadherinEC1-5 reduziert. Durch pharmakologische Hem-mung der Phosphataseaktivität von VE-PTP mittels AKB9778 wird der durch sVE-CadherinEC1-5-induzierte Verlust der Endothelbarriere aufgehoben. Dagegen zeigt die direkte Aktivierung von Tie-2 mittels Angiopoetin-1 keinen protektiven Effekt auf die durch sVE-CadherinEC1-5 gestörte Endothelbarriere. Weitere Analysen zeigen eine erhöh-te Expression von GEF-H1 durch sVE-CadherinEC1-5. Diese ist ebenfalls durch AKB9778 hemmbar.
Zusätzlich zu diesen Untersuchungen wurden die Konstrukte EC1-4 und EC3-5 in ver-schiedene Vektoren kloniert, um zu bestimmen, ob die extrazelluläre Domäne 5 von VE-Cadherin die dominante Rolle bei den sVE-Cadherin-vermittelten Effekten spielt.
Zusammenfassend zeigen diese Untersuchungen zum ersten Mal, dass sVE-CadherinEC1-5 unabhängig von proinflammatorischen Auslösern über die Aktivierung des VE-PTP/RhoA-Signalweges den Zusammenbruch der Endothelbarriere mitversursacht. Dies stellt einen neuen pathophysiologischer Mechanismus dar, der zum Gesamtverständnis der entzündungsinduzierten Barriereveränderungen des Endothels beiträgt.
Adrenocortical carcinoma (ACC) is a rare, but highly aggressive endocrine malignancy. Tumor-related hypercortisolism is present in 60 % of patients and associated with worse outcome. While cancer immunotherapies have revolutionized the treatment of many cancer entities, the results of initial studies of different immune checkpoint inhibitors in ACC were heterogeneous. Up to now, five small clinical trials with a total of 121 patients have been published and demonstrated an objective response in only 17 patients. However, one of the studies, by Raj et al., reported a clinically meaningful disease control rate of 52 % and a median overall survival of almost 25 months suggesting that a subgroup of ACC patients may benefit from immunotherapeutic approaches. Following the hypothesis that some ACCs are characterized by a glucocorticoid-induced T lymphocytes depletion, several studies were performed as part of the presented thesis. First, the immune cell infiltration in a large cohort of 146 ACC specimens was investigated. It was demonstrated for the first time, and against the common assumption, that ACCs were infiltrated not only by FoxP3+ regulatory T cells (49.3 %), but also that a vast majority of tumor samples was infiltrated by CD4+ TH cells (74 %) and CD8+ cytotoxic T cells (84.3 %), albeit the immune cell number varied heterogeneously and was rather low (median: 7.7 CD3+ T cells / high power field, range: 0.1-376). Moreover, the presence of CD3+-, CD4+- and CD8+ ACC-infiltrating lymphocytes was associated with an improved recurrence-free (HR: 0.31 95 % CI 0.11-0.82) and overall survival (HR: 0.47 96 % CI 0.25-0.87). Particularly, patients with tumor-infiltrating CD4+ TH cells without glucocorticoid excess had a significantly longer overall survival compared to patients with T cell-depleted ACC and hypercortisolism (121 vs. 27 months, p = 0.004). Hence, the impact of glucocorticoids might to some extent be responsible for the modest immunogenicity in ACC as hypercortisolism was reversely correlated with the number of CD4+ TH cells. Accordingly, CD3+ T cells co-cultured with steroidogenic NCI-H295R ACC cells demonstrated in vitro an enhanced anti-tumoral cytotoxicity by secreting 747.96 ±225.53 pg/ml IFN-γ in a therapeutically hormone-depleted microenvironment (by incubation with metyrapone), versus only 276.02 ±117.46 pg/ml IFN-γ in a standard environment with glucocorticoid excess.
Other potential biomarkers to predict response to immunotherapies are the immunomodulatory checkpoint molecules, programmed cell death 1 (PD-1) and its ligand PD-L1, since both are targets of antibodies used therapeutically in different cancer entities. In a subcohort of 129 ACCs, expressions of both molecules were heterogeneous (PD-1 17.4 %, range 1-15; PD-L1 24.4 %, range 1 - 90) and rather low. Interestingly, PD-1 expression significantly influenced ACC patients´ overall (HR: 0.21 95 % CI 0.53-0.84) and progression- free survival (HR: 0.30 95 % CI 0.13-0.72) independently of established factors, like ENSAT tumor stage, resection status, Ki67 proliferation index and glucocorticoid excess, while PD-L1 had no impact.
In conclusion, this study provides several potential explanations for the heterogeneous results of the immune checkpoint therapy in advanced ACC. In addition, the establishment of PD-1 as prognostic marker can be easily applied in routine clinical care, because it is nowadays anyway part of a detailed histo-pathological work-up. Furthermore, these results provide the rationale and will pave the way towards a combination therapy using immune checkpoint inhibitors as well as glucocorticoid blockers. This will increase the likelihood of re-activating the immunological anti-tumor potential in ACC. However, this will have to be demonstrated by additional preclinical in vivo experiments and finally in clinical trials with patients.