Obesity impairs virus-specific memory CD8⁺ T cell signalling and function

The main function of the immune system is to protect the host against invading pathogens. Immunological memory is formed after a primary infection and serves to provide protection in a fast and vigorous manner upon secondary encounter with the same pathogen. Under obesity conditions, however, protective immunity is impaired, constituting a significant risk factor for high incidence and severity of re-infections. Indeed, during the current COVID-19 pandemic, obesity has been recognized as a major risk factor for adverse clinical outcomes. Impaired immunity observed in obese individuals has been attributed to a dysfunction in CD8+ T cells, which are essential for the elimination and sterile clearance of viral infections. Yet, the underlying mechanisms of the immune-compromised status of obese individuals remain poorly understood. Using mouse models of diet-induced obesity and lymphocytic choriomeningitis virus infection, we showed increased morbidity and mortality of obese mice after re-infection, recapitulating the clinical situation in humans. Obese mice failed to mount protective immunity and showed a profound loss of virus-specific memory CD8+ T cells in the spleen and liver. This phenotype was associated with a compromised proliferation capacity and reduced ability to produce the effector cytokines IFNg and TNFa. Furthermore, our data revealed a severe decline specifically within hepatic tissue-resident memory T cell pool, which positively correlated with the body weight and contributed to the severe symptoms observed in obese mice. Additionally, we observed enhanced accumulation of IgA+, IL-10 producing, and PD-L1+ B cells in the liver of obese mice, and their absence was associated with normal numbers of hepatic tissue-resident memory T cells. Notably, genetic ablation of IL-10 production by B cells did not reconstitute the memory response, indicating that IgA+ B cells do not exert a suppressive effect towards the virus-specific CD8+ T cells via IL-10. Furthermore, our findings show that the impaired memory response in obese mice is due to T-cell intrinsic mechanisms driven by long-term exposure of virus-specific memory CD8+ T cells to the inflammatory environment, rather than by affecting their development and recruitment. Molecular analysis revealed transcriptional reprogramming of memory CD8+ T cells under obesity conditions, resulting in impaired T-cell receptor signaling. The obesity-inflicted changes in memory CD8+ T cells were highlighted by the impairment of Ca2+ influx upon CD8+ T cell stimulation in vitro. Collectively, our findings indicate that virus-specific memory CD8+ T cells exposed to an obese environment lose their ability to confer protection. The future intention of this project is to identify key molecules within the Ca2+ signalling pathway that could be therapeutically modulated to improve the ability of virus-specific memory CD8+ T cells to provide protection and to improve the immune response of obese individuals to re-infections

Heterogeneity in Kupffer cell-mediated killing of <em>Staphylococcus aureus</em>

Staphylococcus aureus, an opportunistic pathogen, exhibits remarkable adeptness in evading the host's innate immune defenses to establish infections. In this context, Kupffer cells, specialized resident macrophages in the liver, serve as the initial line of defense against S. aureus. However, some of these cells fall short in effectively containing the infection, thereby enabling the escape and widespread dissemination of S. aureus through various evasion tactics. Through in vivo experimentation, we observed that a strategically organized distribution of Kupffer cells, rather than a uniform arrangement along sinusoidal zones, significantly enhances immune response efficacy against systemic bacterial dissemination. Intriguingly, despite the predominant presence of Kupffer cells in periportal regions—aimed at shielding the central vein from pathogenic intrusion—these cells exhibit impaired capability in eradicating S. aureus. Conversely, Kupffer cells positioned in proximity to the central vein and its sinusoids demonstrate heightened proficiency in combatting the pathogen. In-depth analysis through dual-RNAseq of host-pathogen interactions during S. aureus infection unveils a distinctive interplay with the adaptive immune system. Moreover, the central vein microenvironment orchestrates anti-apoptotic pathways, facilitating resilience and cell survival against S. aureus infection and AIM expression and secretion is dependent on the presence of NKT type II. In summary, our findings underscore the pivotal role of spatial zonation in the context of S. aureus infection. Specifically, we highlight the enhanced capabilities of central vein-residing Kupffer cells in managing bacterial escape, potentially attributed to their interaction with type II NKT cells. This interaction, in turn, fosters elevated survival rates and augments the arsenal of antibacterial defense mechanisms

FVIII-albumin fusion proteins enhance the induction of immune tolerance towards coagulation factor VIII in haemophilia A mice

Haemophilia A (HemA) is an X-linked recessive disease caused by the lack of functional coagulation factor VIII (FVIII) causing life-threatening prolonged bleeding events due to a coagulation incapacity. HemA is treated by prophylactic or on-demand infusions of recombinant FVIII (rFVIII), according to the grade of disease severity. Individuals affected by severe haemophilia A are characterised by large deletions or intron inversions in the fviii gene and therefore present a significantly different portion of FVIII compared with the wildtype protein present in healthy individuals. Therefore, intravenously-injected exogenous rFVIII is recognised as a foreign antigen. FVIII-specific B cells are activated upon immunisation and trigger the production of anti-drug neutralising antibodies, known as inhibitors. Presently, the only known method to induce tolerance towards FVIII is the Immune Tolerance Induction regime, known as ITI, which consist of repetitive high-dose FVIII infusions pursued until the inhibitor levels are abrogated. The limitations and poor accessibility of the ITI therapy open the door to alternative strategies to enhance tolerance induction and improve the quality of the individuals’ life. This work aimed to induce immune tolerance towards exogenous FVIII by the administration of FVIII-albumin conjugates in a haemophilia A mouse model. Here, FVIII-albumin fusion proteins were generated by chemical cross-linking of FVIII to albumin in a 1:1 or 1:5 molar ratio, referred to as FP(1:1) and FP(1:5), respectively. Unlike the ITI models, the fusion proteins were injected intravenously in singular weekly intervals and aligned with FVIII control group. The mechanisms underlying the FVIII-specific immune response were elucidated in vivo by flow cytometry, confocal microscopy and ELISA and by the establishment of an in vitro HemA splenocytes cultures. Collectively, the data indicated that FVIII-albumin impairs the development of FVIII-specific B cells into activated subtypes reducing dramatically the inhibitor production compared with FVIII alone, while being coagulative active. Fusion protein is transported to the liver where it could be internalised by professional antigen-processing cells, including dendritic cells and potentially by macrophages. Nevertheless, fusion protein treatments affect the co-stimulation of APCs causing the disruption of follicular CD4+ T helper cell activation. CD25+ regulatory T cells may contribute to the suppression of epitope-specific T helper cells. In vitro co-cultures indicated that the Fas/FasL and PD-1/PD-L1 axes may be employed as a potential FVIII-specific B cell suppressive Treg-mediate mechanism. Overall, FVIII-albumin stimulation in vitro failed to stimulate FVIIIspecific B cells and TFH in comparison with FVIII stimulation. To explore the membrane trafficking of albumin and FVIII-albumin, an uptake assay was established in RAW264.7 macrophages. Here, I evaluated the compartmentalisation of albumin within the recycling or degrading endosomes and its segregation into recycling tubules by confocal microscopy and live cell imaging. FVIII-albumin fusion protein, similar to albumin alone, segregated from FVIII-positive endosomes into tubular structures which may undergo membrane trafficking and recycling. Overall, FVIII-albumin fusion proteins seem to be a promising therapeutic tool to enhance induction of immune tolerance protocols towards FVIII

Morphologic adaptations and hydrodynamics of 'pelvic brooding', a derived reproductive strategy in Sulawesi ricefishes (Beloniformes: Adrianichthyidae)

(noch nicht zugänglich / not yet accessible

Investigation of the Lysosomal Proteome and the Phosphorylation Dynamics of Transcription Factor EB Using Mass Spectrometry

(noch nicht zugänglich / not yet accessible

Recognition of orthoflaviviral RNA in mosquitoes and viral countermeasures

Various infectious diseases are caused by mosquito-borne orthoflaviviruses affecting people worldwide. Efficient transmission of these viruses highly depends on the pathogen’s ability to overcome the host immune system. Previous studies showed that orthoflaviviruses evade the vertebrate immune system by capping their viral genome via a cap-N1-2’-O-methyltransferase encoded in the non-structural protein NS5. This thesis aimed to analyze whether orthoflaviviruses also escape the insect immune system by modulating their 5’ cap structure via 2’-O-methylation. Using the yellow fever virus (YFV) vaccine strain (YFV-17D cap1) and a methyltransferase deficient mutant (YFV-17D cap0), this thesis demonstrates that the replication of YFV-17D cap0 is also impaired in mosquito cells. Since YFV-17D is unable to replicate in mosquitoes, a YFV-Asibi cDNA clone was additionally established to compare the respective cap1 and cap0 variants in vitro and in vivo. Similar to YFV-17D cap0, YFV-Asibi cap0 was suppressed in mosquito cells in a Dicer-2 independent manner but to a slightly lower extent. These data indicate that YFV-Asibi counteracts the insect antiviral discrimination mechanism of cap0 RNA. Studies comparing chimeras between YFV-Asibi and YFV-17D aimed to pinpoint the viral genes responsible for counteracting the discrimination mechanism. The results suggest that a synergy of several non-structural proteins is involved in this mechanism. In the case of YFV, the counteraction seems to be linked to a faster viral replication at early time points. Furthermore, after oral infection of Aedes aegypti mosquitoes, YFV-Asibi cap1 replicated in the mosquito midgut and secondary tissues like legs plus wings. Conversely, replication of YFV-Asibi cap0 was suppressed in the midgut and nearly blocked in secondary tissues. Intriguingly, efficient replication of YFV-Asibi cap0 occurred after intrathoracic infection, indicating the existence of a potential receptor or protein discriminating between cap1 and cap0 RNAs in the midgut or the midgut barrier. Since the methyltransferase is highly conserved between orthoflaviviruses, DENV cap0 was established as well. Comparative growth curve kinetics revealed that DENV cap0 replication is not suppressed in mosquito cells, implying that DENV also counteracts the insect antiviral discrimination mechanism of cap0 RNA but more strongly than YFV-Asibi. In summary, the obtained results suggest the existence of an innate 5’ RNA-modification recognizing effector protein in mosquito cells and mosquitoes. Further, orthoflaviviruses counteract this effector protein by different means, leading to different levels of cap0 virus replication

Modulation of filamin C interactions and function by mechanical stress-induced phosphorylations

(noch nicht zugänglich / not yet accessible

Regulation of FVIII-specific immunity in Hemophilia A mice

The major complication of Hemophilia A treatment is the formation of inhibitors that neutralize the administered FVIII. Immune tolerance induction (ITI) is the most frequently used therapy in order to establish long-lasting tolerance towards FVIII in patients with inhibitors. However, predicting ITI success is challenging and the underlying cellular and molecular mechanisms are poorly described, thus therapy stability and duration is individualized, and might be influenced by several risk factors. Here, I demonstrate that upon successful tolerization of HemA mice, a PR8 infection in the lung triggered a systemic alteration of the inflammatory state. FVIII-specific B cells, responsible for the anti-FVIII humoral alloimmune response, are increased upon infection, while the FVIII-specific Tregs and their PD-L1 expression, induced during ITI in vivo, were reduced. Additionally, the FVIII-specific T cell compartment changed into a pro-inflammatory phenotype with an increase of FVIII-specific Th17 and Tfh cells. The inter-organ crosstalk between lung and spleen, was mediated by IFN-g, reactivating FVIII-specific B cells, which exhibited a significantly elevated IFN-gR expression. Consequently, these FVIII-specific B cells shifted their phenotype, by increasing their CD80 expression and an initiated IL-6 production. This shift contributed to a loss of the previously established tolerogenic environment towards FVIII achieved through the ITI protocol by fostering T cell dedifferentiation upon IL-6 exposure. Finally emphasizing the importance of the IL-6 signaling in tolerance disruption, the inhibition of IL-6R proved that the tolerized phenotype was restored upon application. Furthermore, other external elements such a high salt diet (HSD) did not impact the ITI outcome. Nevertheless, the HSD in wildtype mice induced elevated corticosterone levels in the bloodstream which modified the uptake behavior of splenic red pulp macrophages and seemed to promote a shift towards a pro-inflammatory phenotype. This was accompanied by a reduction of the FVIII levels in the bloodstream, disturbing the equilibrium of the FVIII homeostasis. Overall, these results may pave the wave for novel therapeutic intervention strategies to improve ITI success and the hemostatic FVIII abundance through a more detailed understanding of the interplay between FVIII, immune and tolerance responses and external factors

Perturbative and non-perturbative theories of structure formation

(noch nicht zugänglich / not yet accessible

Total Synthesis of Farnesyl Lipid I/II Analogues and Contributions to the Total Synthesis of Neaumycin B

(noch nicht zugänglich / not yet accessible