Die Rolle von Virulenz-assoziierten Proteinen in der Wirtsspezifität von <em>Rhodococcus equi</em>

Rhodococcus equi ist ein Pathogen von jungen Pferdefohlen und immunsupprimierten Menschen. R. equi-Isolate aus verschiedenen Organismen enthalten verschiedene Virulenzplasmide. Als eine zentrale Virulenzeigenschaft kann R. equi die Reifung der es umgebenden Phagosomen in Makrophagen hemmen. In der vorliegenden Arbeit wurde untersucht, welchen Einfluss verschiedene R. equi-Virulenzplasmide auf die Infektion verschiedener Säugerzellen haben. Die Resultate geben Einsicht in die Mechanismen, mit denen R. equi verschiedene Säugerspezies infizieren kann

Untersuchung der molekularen Interaktion endogener und synthetischer RNA mit dem zytosolischen Immunrezeptor RIG-I

Die Helikase RIG-I spielt als zytosolischer Immunrezeptor eine zentrale Rolle bei der Abwehr der meisten hoch pathogenen RNA-Viren. RIG-I erkennt 5’triphosphorylierte doppelsträngige RNA (3P-dsRNA), wie sie bei der Replikation von Viren entsteht und initiiert durch Ausschüttung von Typ I Interferonen und Chemokinen eine antivirale Immunantwort. Bisherige Publikationen fokussierten lediglich die Struktur des RIG-I Liganden. Basierend auf kristallographischen Studien wurden mit 3P-dsRNA interagierende Aminosäuren der RIG-I–RNA-Bindedomäne (RD) identifiziert und deren biologische Relevanz in dieser Arbeit durch Mutationsanalysen evaluiert. Es konnte gezeigt werden, dass die Bindung von α- und β-Phosphat durch jeweils 2 basische Aminosäuren essentiell ist, wobei die Erkennung des γ-Phosphates lediglich für die RIG-I-Aktivierung bei niedrigen Ligandenkonzentrationen notwendig wird. Als zusätzlich obligatorische Kontakte für RIG-I-Aktivierung ergaben sich die Stacking-Interaktion einer aromatischen Aminosäure mit dem 5’ basengepaarten RNA-Terminus und die Bindung eines Internukleotidphosphates des RNA-Rückgrates. In der Kristallstudie konnte zusätzlich die hoch konservierte mit 3P-dsRNA interagierende Aminosäure His830 identifiziert werden. Ihr konnte eine immunregulatorische Rolle zugeordnet werden: endogene mRNA besitzt an ihrem 5’-Ende ein Triphosphat, das durch ein m7G-Cap modifiziert ist. Es konnte gezeigt werden, dass nicht, wie bisher angenommen, das m7G-Cap allein, sondern erst die für Vertebraten charakteristische 2’-OCH3-Gruppe am ersten Ribonukleotid (N1) endogene RNA immunologisch inert macht. Diese Immuntoleranz von endogener RNA mit 2’-OCH3-Gruppe an N1 wird durch His830 vermittelt, dessen sperrige Seitenkette die Interaktion 2’-modifizierter RNA mit RIG-I inhibiert. Untersuchungen an Gelbfieber-Virus ergaben, dass auch Viren diesen Mechanismus nutzen: verlieren sie die Kompetenz zur N1-2’-O-Methylierung, so erschwert dies kaum die Virusreplikation sondern resultiert in einer verstärkten RIG-I-abhängigen Immunantwort. Insgesamt konnten in dieser Arbeit damit sowohl neue Erkenntnisse in der RIG-I-Liganden-Interaktion auf struktureller Ebene erlangt, als auch ein neuer Toleranzmechanismus von RIG-I für endogene RNAs aufgedeckt werden. Zudem konnte geklärt werden, dass eine RIG-I-Aktivierung auch ohne Triphosphorylierung möglich ist, dabei aber niedrig affin abläuft und eine Sequenzspezifität auftritt. Diese neuen Erkenntnisse zusammen können in Zukunft genutzt werden um ein Molekül zu entwickeln, das eine gezielte kurativ Immunantwort in verschiedenen Tumorentitäten initiiert

<em>Salmonella</em>-specific CD4⁺ T cells in the murine model of Salmonellosis

Salmonella infection is a major cause of morbidity and mortality worldwide, which requires a deeper comprehension of the protective immune mechanisms, in order to develop more effective vaccines. While the importance of CD4+ T cells during Salmonellosis is well established, the specific antigenic targets, in vivo dynamics and phenotype of Salmonella-specific CD4+ T cells remain incompletely understood. In this project, CD4+ T cell antigen targets were elucidated from a novel Salmonella-derived MHC class II immunopeptidome and their immunogenicity was validated in a murine model of Salmonellosis. Four antigens were selected for generating peptide:MHC class II tetramers. These tetramers facilitated the direct identification, enumeration and characterization of previously unreported Salmonella-specific CD4+ T cells during murine Salmonellosis. The selected antigenic targets varied in their immunogenicity as well as subcellular localization, expression levels and conservation of their source proteins, aimed at representing T cell responses to diverse antigens. Remarkably, in some but not all animals, analysis of tetramer and TCRß staining revealed at least two distinct populations of specific CD4+ T cells, prompting investigation into TCR usage by single-cell RNA sequencing. This approach shed light on the reasons behind the existence of multiple populations and provided insights into the polyclonallity of Salmonella-specific CD4+ T cell responses. Moreover, Salmonella-specific CD4+ T cells exhibited a T helper 1 effector phenotype with surface marker expression consistent with homing to the liver, a critical site of Salmonella infection. Intriguingly, Salmonella-specific CD4+ T cells were readily available for reamplification upon challenge with wild type Salmonella, if animals were previously vaccinated with an attenuated Salmonella strain, suggesting that the selected peptides may have vaccine potential. Additionally, Salmonella overexpression mutants were generated, increasing the expression of one of the source proteins of the antigenic targets. When these mutants were used for infection experiments, the frequency of specific CD4+ T cells was increased, as did the frequency when multiple populations stained with a single tetramer were identified, without affecting the Salmonella-induced gross pathology. This approach offers new avenues for understanding the dynamics of Salmonella-specific CD4+ T cell responses and their modulation for therapeutic interventions. Our results demonstrate the feasibility and utility of using immunopeptidomics and tetramer technology to study Salmonella-specific CD4+ T cell immunity in a murine model of Salmonellosis. Furthermore, the novel antigens and tetramers provide valuable tools for understanding the mechanism of protective immunity, paving the way for the urgently needed development of next-generation vaccine formulations against Salmonella infections

The Role of Muscularis Macrophages in Mediating Local and Systemic Immune Responses in a Mouse Model of Multiple Sclerosis

Multiple sclerosis (MS) is a chronic neurodegenerative disease, likely of autoimmune origin, which mainly affects the central nervous system (CNS). It is often associated with impaired gut motility, causing symptoms like constipation, diarrhea or faecal incontinence in most patients with MS. Recently, it was identified that the enteric nervous system (ENS), the intrinsic nervous system of the gastrointestinal tract, which regulates motility and secretion, is an additional autoimmune target in MS and its animal model, experimental autoimmune encephalomyelitis (EAE). Muscularis macrophages (MMs) are located in close proximity to enteric neurons. Both regulate each other by secreting survival factors that are essential for the function of the other population. MMs are known to be regulators of the local immune response in a variety of pathological conditions, such as post-operative ileus and intestinal infection with bacteria or helminths. Additionally, MM depletion was shown to impair gut motility. In this project, we investigated the involvement of MMs in the pathogenesis of EAE. To this end, we performed single-cell RNA sequencing and identified that MMs upregulate anti-inflammatory and neuron-associated genes. Furthermore, we established a model to combine EAE with MM depletion. We observed an exacerbated clinical outcome in MM-depleted mice that was associated with an earlier and increased infiltration of immune cells into the CNS, leading to a more severe axonal damage. Using high-dimensional flow cytometry, we identified that gut-associated effector T cells are activated earlier in MM-depleted animals due to an increase in the number of dendritic cells in the muscularis externa. Our results suggest an increased pathogenicity end encephalitogenicity of type 17 helper T cells that impair the blood brain barrier integrity and allow an early infiltration of immune cells into the CNS. Taken together, our study identifies MMs to be an essential regulator of the systemic immune response that originates in the gut and to display a novel target for potential MS therapies

Modulation of the immune response after myocardial infarction to improve cardiomyocyte engraftment : Inhibition of the NLRP3 inflammasome enhances cell engraftment and preserves cardiac pump function

The decline in cardiac pump function due to the formation of a fibrotic scar is one of the most severe consequences of myocardial infarction (MI) and can ultimately result in terminal pump failure of the organ. Currently available therapies primarily target the remaining viable myocardium and aim to reduce myocardial workload but do not directly treat the infarct scar. Re-population of the scar area with transplanted cardiomyocytes is therefore an attractive therapeutic strategy, However, previous studies have demonstrated that the majority of injected cardiomyocytes are lost very early after delivery into the freshly infarcted myocardium. Loading of cells with magnetic nanoparticles (MNP) prior to intramyocardial injection in the presence of an external magnetic field has emerged as a promising approach to enhance cellular retention within the tissue, yet a substantial loss of transplanted cells over time has still been observed. The NLRP3 inflammasome is a multi-protein complex that assembles in response to endogenous signals. Its terminal effector, caspase 1, mediates the maturation of interleukin 1β (IL-1β), a potent pro-inflammatory cytokine, that plays a key role in initiating the post-MI immune response. Pharmacological inhibition using the NLRP3 specific inhibitor MCC950 has been shown in animal models to improve cardiac pump function after MI and to preserve graft function following heart transplantation. On the basis of these findings, the present study investigated whether NLRP3 inflammasome inhibition can enhance short- and long-term engraftment of transplanted embryonic cardiomyocytes (eCM). In a mouse model of permanent coronary ligation (LADx), 2 × 105 MNP-loaded eCM were injected intramyocardially under the influence of an external magnetic field. Additionally, animals received MCC950 intraoperatively and every other day until day 14 post-MI. In the absence of NLRP3 inhibition, only 1.1% of the injected cells remained within the scar 14 days after transplantation, whereas MCC950 treatment significantly increased this proportion to 5.5%. At this time point, mice treated with the combination of eCM and MCC950 showed an improvement in left ventricular ejection fraction (EF), which was not observed in animals receiving eCM alone. Interestingly, MCC950 monotherapy also led to an improvement in EF. Infarct scar volumes did not differ between groups; however, no transmural lesions were detected in MCC950-treated mice 14 days post-MI. Histological analyses confirmed a reduced number of dying cells in the infarct area on day 1 post-MI and a decrease in leukocyte and fibroblast proliferation on day 3 post-MI in MCC950 treated animals. The efficacy of MCC950 treatment was further supported by reduced expression of NLRP3 and IL-1β on day 3 post MI. Eight weeks after MCC950 treatment, the number of engrafted eCM remained 4.3-fold higher compared to animals without NLRP3 inhibition, although a loss of approximately 75% of the cells relative to the 2 week time point was still observed. The beneficial effect of MCC950 on EF persisted long term, both in animals receiving cell transplantation and in those treated with MCC950 alone. In summary, MCC950 attenuates the post-infarction immune response and improves both short- and long-term engraftment of intramyocardially transplanted eCM. Moreover, cardiomyocyte transplantation and MCC950 treatment, either in combination or as standalone interventions, enhance cardiac pump function following MI

Dopamine dynamics in the basolateral amygdala during associative learning and exploration

Dopamine release in the basolateral amygdala (BLA) is thought to contribute to salience attribution and associative learning; yet its role during self-paced behaviours has remained unexplored. Using fibre photometry combined with the genetically encoded dopamine sensor dLight1.1, I monitored real-time dopamine dynamics in freely moving mice during discriminative fear conditioning, extinction, and multiple exploratory paradigms. By aligning signals to both sensory cues and pose-estimated behaviours, I show that BLA dopamine transients are evoked not only by aversive unconditioned (US) and conditioned stimuli (CS+), but also during self-paced exploratory behaviours such as open arm entries, head dips, and rearing. In contrast, dopamine release was suppressed during non-exploratory or defensive states. Critically, BLA dopamine release was independent of speed, indicating a state-reflective rather than a movement-driven signal. During target-directed exploration (i.e., object or social stimulus), I implemented a vector-based classification of body orientation and proximity, which revealed that dopamine release in the BLA scales with both proximity to and orientation towards the target. BLA dopamine was differentially modulated by stimulus identity, with proximity to objects evoking a stronger dopamine release compared to proximity to social targets. Sensory cues and behavioural states were hierarchically ranked by signal magnitude, with stronger dopamine signals for highly salient cues (e.g., US and CS+), intermediate for exploratory states, and suppressed during non-exploratory/defensive states. Notably, dopamine release during the predictive CS+ persisted in high fear state and decreased upon extinction, consistent with salience revaluation and an expectation-modulated teaching signal. Together, these results show that BLA dopamine can dynamically weigh external cues and internal states to guide adaptive behaviour. This function has broad implications for grasping how BLA dopamine may support flexible learning and state regulation, and how its dysregulation may contribute to psychiatric disorders such as post traumatic stress disorder (PTSD) and anxiety

Elucidation of the neuroanatomical basis of fine motor function across lifespan

Fine motor function, the coordinated use of a large number of relatively small arm and hand muscles, is indispensable for performing daily activities and serves as a critical indicator of neurological health. Its decline often accompanies the onset of neurodegenerative diseases like stroke or PD and may even predict cognitive impairment. However, the specific neurobiological pathways linking these together remain poorly understood. A comprehensive understanding requires investigations on how fine motor performance relates to brain structures, white matter health, and disruptions within brain networks that are believed to underpin the parallel decline in both motor and cognitive performances. This critical knowledge gap persists as comprehensive, population-based studies have been lacking. To address this gap, we used cross-sectional and longitudinal data from the Rhineland Study, a large-scale population-based cohort study. A digital spiral drawing test was utilized to evaluate fine motor skills from three aspects: tracing precision (deviation area), tracing velocity, and frequency of tremor. Brain volumetric and cortical thickness measures were obtained from 3T T1 MRI scans. WMH were obtained from T1, T2 and T2 FLAIR sequences, while white matter integrity was derived from diffusion weighted imaging. Brain functional connectivity was assessed using resting-state functional MRI. Standardized cognitive domain scores were derived from extensive neuropsychological test batteries. Leveraging data from 8,317 participants of the Rhineland Study, our investigation revealed a multi-faceted relationship between fine motor function and brain health. Firstly, our findings indicate that fine motor function is worse in older adults, and is better in women. Moreover, changes in total brain volume and the thickness of several key motor cortices are robustly related to fine motor function, with the strongest effect for tracing precision. Second, our findings indicate that while a larger WMH burden is associated with worse fine motor control, also changes in the NAWM microstructural integrity of motor and association fibers affect fine motor function. Finally, our findings indicate that fine motor function measured by digital spiral drawing test provides a sensitive, non-invasive and easily administered marker of early cognitive decline and functional brain network disruption, which could be leveraged to identify individuals at increased risk of neurodegenerative diseases. In summary, this thesis investigated the neuroanatomical basis of fine motor function across lifespan, and explored their associations with cognitive function, using multimodal neuroimaging and large-scale population-based data

Alterations of the nutrient uptake depth as inferred from radiogenic strontium isotopes in two case studies : a forest ecosystem and an agricultural field

Climate change and its associated effects, such as higher temperatures and atmospheric CO2 concentration, pose increasing challenges to both forest and agricultural ecosystems. Nutrient uptake from deeper soil might be a promising adaptation strategy of the plants, as the subsoil contains additional water and nutrient resources. Yet, we know little on how forests adapt their nutrient acquisition strategies to different climate conditions, or how the nutrient uptake depth of crops can be manipulated by different management strategies, particularly for sandy soil. My thesis aimed to 1) investigate whether and how Norway spruce responds to global changes by changing its mean nutrient uptake depth, and 2) to evaluate the role of subsoil management for the nutrient uptake from sandy soils. To address these objectives, radiogenic strontium isotope ratios (87Sr/86Sr) were used as a geochemical proxy to trace nutrient sources and uptake depth in forest and agricultural systems. In a long-term forest monitoring site at Mitterfels forest (Bavaria, Germany), I tracked changes in needle biomass, nutrient concentrations and 87Sr/86Sr ratios in Norway spruce (Picea abies L.) from 1992 to 2014. In an agricultural experiment trial on sandy soil (Retisol) at Thyrow (near Berlin, Germany), I investigated the effects of subsoil management strategies including strip-wise deep loosening with and without the incorporation of biowaste compost on nutrient uptake depth in crops. Needle biomass significantly increased over the decades, indicating enhanced plant growth under elevated CO2. Nutrient concentrations of K and P declined, but Mg largely increased by ~ 40%, often beyond requirement levels. Simultaneously, the 87Sr/86Sr ratios in spruce needles increased from 0.7147 to 0.7177, suggesting a shift in nutrient uptake toward older organic layers and deeper mineral soil horizons with higher 87Sr/86Sr ratios. These findings indicate that spruce trees adapted to climate change by deepening their nutrient uptake depth to satisfy increased nutrient demand. The nutrient status and 87Sr/86Sr ratios in the arable subsoil were largely altered by the additional nutrients from composts compared to the control and deep loosening plots; however, Sr isotope ratios in crop tissues remained unchanged. Hence, the crop's primary nutrient source remained in the topsoil despite the elevated nutrient levels in the subsoil. These findings underscore the importance of soil water retention in sustaining and improving crop performance in the sandy soil. Overall, my data demonstrates that radiogenic strontium isotopes provide a valuable approach to identify plant nutrient sources and trace nutrient uptake depth in different ecosystems. Both systems illustrate how plants regulate nutrient uptake depth in response to environmental drivers such as climate change or agricultural management, highlighting the interaction between plant plasticity and external environment changes in controlling nutrient acquisition from varying soil depths

Cost-Effectiveness of Protective Equipment Stockpiles for Reducing Essential Worker Absenteeism in a Pandemic

Pandemics with respiratory pathogens in a globally connected world will remain a threat to critical infrastructure and society, making it crucial to provide essential workers with protective equipment quickly. This study explores the cost-effectiveness of two different strategies: 95 % efficacy single-use respirators versus reusable respirators with 99 % efficacy. I estimated the costs per absentee day averted across three pandemic scenarios: Influenza, Covid-19, and Disease X. I measured direct absenteeism with a modified SIR model and assumed the respiratory protective equipment’s filtration efficacy as the reduced chance of infection per encounter (protectiveness). I used behavioural modelling for indirect absenteeism. For each disease scenario, I compared two respiratory protective equipment stockpiling strategies in terms of their costs and protectiveness for essential workers. In all three scenarios, reusable and highly protective equipment was found to be more cost-effective than single-use, yielding €5.77 (single-use) and €3.78 (reusable) for influenza, €5.76 and €3.58 for Covid-19, and €9.72 vs €5.13 for Disease X. The more severe the pandemic scenario in death count and infections, the higher the difference in cost-effectiveness between the two stockpiling strategies (34.5 %, 37.9 %, and 47.2 %). My model suggests that keeping a reserve of reusable, highly protective elastomeric respirators might improve worker attendance during severe pandemics. This underscores the need for pathogen-agnostic research and the inclusion of essential workers beyond the healthcare sector in pandemic planning (such as agriculture, water and electricity maintenance, law enforcement) in preparing for yet unidentified pandemic pathogens. However, this study has certain limitations. The model assumes that filtration efficacy directly equates to protection, without accounting for factors such as proper fit, user compliance, and training, which significantly impact effectiveness in real-world settings. Additionally, the cost analysis focuses on procurement and storage, excluding operational expenses like distribution and disposal, which may affect the overall cost-effectiveness. Ultimately, stockpiling reusable respirators might still be a cost-effective strategy to maintain essential workforce operations and enhance pandemic resilience

ADAR1 deficiency in regulatory T cells leads to MDA5 and PKR dependent systemic autoimmunity in mice

Type I interferon (IFN-I) production, triggered by innate sensors upon recognition of viral nucleic acids, is essential for antiviral host defense. Mutations in proteins involved in nucleic acid metabolism, sensing and downstream signaling, cause rare monogenic diseases termed type I interferonopathies, which are characterized by chronic increased production of IFN-I and expression of IFN stimulated genes (ISGs), and a variety of inflammatory and autoimmune phenotypes. Gain-of-function (GOF) mutations in IFIH1, encoding the dsRNA sensor MDA5, and loss-of-function (LOF) mutations in ADAR1 (ADAR1), which by adenosine-to-inosine editing of dsRNA prevents their aberrant sensing by MDA5, are associated with the interferonopathy Aicardi–Goutières syndrome (AGS). Previous findings showed that expression of constitutively active MDA5 in regulatory T cells (Tregs) – crucial in preventing autoimmunity, led to autoimmune phenotypes in mice. It was therefore of interest if ADAR1 deficiency in Tregs (Foxp3∆Adar1), leading to aberrant MDA5 activation, would also be detrimental. Indeed, Foxp3∆Adar1 mice, exhibited Treg depletion, leading to lethal autoimmunity and autoinflammation. The involvement of pathways downstream of ADAR1 in Foxp3∆Adar1 phenotypes, was investigated by genetic ablation of MAVS (the signaling adaptor of MDA5), PKR and ZBP1 – two other innate sensors and ISGs activated upon ADAR1 deficiency. MAVS knockout improved inflammatory phenotypes, but did not prevent Treg depletion. Moreover, in vivo inhibition of the IFN-I receptor (IFNAR) before phenotypic onset, prolonged Mavs−/−Foxp3ΔAdar1 survival, indicating enhanced IFNAR signaling independent of MDA5–MAVS. While ZBP1 knockout had minor effects, PKR knockout prevented Treg depletion – highlighting its involvement in Foxp3∆Adar1 phenotypes. Accordingly, MAVS and PKR double-knockout fully rescued Foxp3∆Adar1 mice. Notably, patients with AGS carrying mutations in ADAR1 and IFIH1, exhibited reduced percentages of highly suppressive effector Tregs, markedly associated with ADAR1 LOF. While supported by evidence in mice, this finding is limited by human non-Tregs that inexplicably express the Treg-integral protein FOXP3. Nevertheless, it would be valuable to understand how Tregs are affected by interferonopathy-causing mutations, to examine their potential role in the pathogenesis of these diseases, and explore methods that enhance Treg function to ameliorate autoimmune symptoms