Microbiology & Immunology - Theses
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Evolutionary Dynamics of Successful Clones of Methicillin-Resistant Staphylococcus aureus in Australia and New Zealand
Staphylococcus aureus is a common cause of bacterial infections in humans and a leading nosocomial pathogen, associated with significant morbidity, mortality and economic impact. A cornerstone in the evolution of staphylococcal lineages that infect humans has been their remarkable ability to rapidly and efficiently develop or acquire mechanisms of antimicrobial resistance, impacting effective disease management, prevention, and eradication. Improving these measures or developing novel approaches requires a comprehensive understanding of the infecting agent and while S. aureus has been extensively studied there remains considerable gaps in our knowledge surrounding this pathogen, especially concerning population dynamics. What drives the emergence and/or persistence of certain staphylococcal lineages? What evolutionary pathways and molecular mechanisms are being utilised and under what circumstances? What environmental and host factors have the greatness influence on bacterial population adaptation? And ultimately, what are the consequence of these population level changes and what impact do these have on staphylococcal disease? This thesis represents three projects undertaken to strengthen our understanding of the evolutionary dynamics surrounding staphylococcal population adaptation, using a combination of comparative genomics and detailed phenotypic profiling to provide insight into antibiotic-resistant lineages of S. aureus that circulate in Australia and New Zealand. The first project investigated the long-term persistence of the globally disseminated, multidrug resistant hospital associated methicillin-resistant S. aureus (MRSA) lineage ST239 in Australia. From this work, it has been identified that the ST239 MRSA population circulating in Australia represents not one, but two genetically distinct clades; the previously unrecognised introduction of the Asian-Australian clade followed by successful local expansion in the state of Victoria has contributed to the persistence of ST239 by supplementing the diminishing population size of the local clone, the Australian clade. The ability of the Asian-Australian clade to spread following its introduction is owed to the reduced susceptibility this population developed against anti MRSA antibiotics, namely the glycopeptides and daptomycin, prior to its importation. This same phenotype has convergently emerged in the local Australian clade. However, this adaption has come at a cost as both clades were found to have reduced replicative fitness and impaired pathogenic potential, the latter occurring through loss of functionality or reduced expression of a staphylococcal global virulence regulatory system, the accessory gene regulator. The second project considered a different evolutionary circumstance; the rapid emergence of a novel MRSA lineage in the New Zealand community. This region has a high incidence of staphylococcal skin and soft tissue infection and over the last two decades has seen a significant shift in the local molecular epidemiology of S. aureus, with the emergence of multiple fusidic acid resistant clones. This work has focused on the predominant MRSA lineage, an ST5 clone locally referred to as AK3. Genomic investigation of this lineage has found that AK3 represents a single phylogenetic clade that has arisen through local population expansion, having emerged from a resident fusidic acid susceptible methicillin-susceptible S. aureus upon the acquisition of a novel chimeric SCCmecIVa-fusC II mobile element harbouring the fusidic acid resistance determinant fusC. This phenomenon was not restricted to ST5, with the other newly emerged lineages having acquired fusC via structurally distinct SCC and SCCmec elements. Indicating that, the unregulated use of fusidic acid in the region has supported the emergence and expansion of these novel lineages and is potentially contributing to the high local incidence of staphylococcal infection. Mobile elements can greatly influence staphylococcal populations, therefore the third project focused on exploring the evolution of the multidrug resistant plasmid family pSK1 and the role it has played in augmenting antimicrobial resistance and biocide tolerance in the ST239 MRSA population in Australia. Modelling the evolutionary history of this plasmid identified that it emerged during the late 1970s and over the following four decades has undergone significant structural change, involving a combination of chromosomal integration, transposon loss/gain, structural inversion and deletion events. When aligned to a phylogenetic model of the ST239 population, it became apparent that these changes in plasmid configuration represented a clear pathway of step wise adaptation; the shortened, chromosomally integrated plasmid structural variants having emerged on multiple occasions in a convergent manner. Further, these changes correlated with the development of enhanced tolerance against chlorhexidine, an important finding as it implicates biocide use as a factor potentially influencing MRSA evolution. Collectively, this work enhances our understanding of how antibiotic-resistant lineages of S. aureus evolve and adapt; exemplifying the evolutionary pathways facilitating adaptation in staphylococcal populations and the circumstances under which they are used. Further, it provides detailed insight into two highly successful lineages of MRSA. This knowledge can be exploited to improve measures that reduce the burden of antibiotic-resistant staphylococcal infection. Importantly, this work enhances and reinforces our awareness about the consequences of antimicrobial overuse and misuse.
MAIT cell diversity, function and impact on dendritic cells
T cells represent an important component of the immune system. Whilst early studies were largely focused on the role of conventional CD8+ and CD4+ T cells that recognize peptide-antigens in association with MCH molecules, more recently, T cells that recognize other types of antigens have been described. Mucosal associated invariant T (MAIT) cells are such a cell population and belong to the broad family known as ‘unconventional’ T cells, due to their non-peptidic antigen recognition characteristics. MAIT cells are defined by their recognition of microbial vitamin B2 metabolites presented by MHC related protein 1 (MR1). Upon antigen recognition they immediately display effector functions, like secreting cytokines and expression of cytotoxic proteins. Whilst the majority of MAIT cell studies have focused on the role of MAIT cells to bacterial infections, however their function within the immune system and interaction with other immune cells is still unknown. This thesis focuses on the role that MAIT cell activation has on other immune cells like dendritic cells (DCs) and other T cells. Furthermore, the full potential of MR1-recognition by other T cell subsets was also examined, revealing that MR1-reactive T cells may extend beyond what is currently describe as MAIT cells. The first chapter of this thesis investigates the role of MAIT cell activation on DCs in an in vivo mouse model. MAIT cells were activated by intratracheal injection of the activating MAIT cell antigen 5-amino-6-D-ribitulaminouracil/ methylglyoxal (5-A-RU/MeG). This activation of MAIT cells led to migration of DCs from the lung to the mediastinal lymph node (medLN) as well as DC maturation in an MR1-dependent manner. Furthermore, production of the chemokines CCL17 and CCL22 was induced by MAIT cell activation, which suggests that MAIT cells are able to modulate the immune system far more than previously thought. The possible role of MAIT cell induced DC maturation on initiation of a CD8+ T cell response is analyzed within the second result chapter. No enhanced antigen-specific CD8+ T cell response to the model antigen ovalbumin (OVA) was observed by additional MAIT cell activation. Besides MAIT cells, recently more MR1-reactive T cells were identified. By using antigen-loaded MR1 tetramers, a population of FOXP3+ T-bet+ T cells was identified in human thymus that can bind to MR1 tetramers. In the third chapter this FOXP3+ T-bet+ T cell population was further characterized by analysis of their phenotype as well as their TCR usage. The results in this chapter will serve as a basis for further investigation of the diversity of MR1-recognition within the T cell pool. In conclusion, this thesis reveals a new role of MAIT cells that may be used to manipulate their functions to treat different diseases like autoimmune diseases or cancer. Moreover, the knowledge of MR1-reactive T cell diversity is extended including a potential regulatory role of MR1-reactive T cells and MAIT cells. In summary, this thesis extends the current knowledge of MAIT cell biology.
Role of the chemokines CCL17 and CCL22 in the immune defence against Salmonella infection
The chemokines CCL17 and CCL22 are both ligands of the chemokine receptor CCR4, which is expressed on dendritic cells (DC) and a variety of different effector T cells including regulatory T cells (Treg). Both chemokines are mainly produced by DC, but also by macrophages. CCL17 promotes numerous inflammatory and allergic diseases, whereas CCL22 is rather associated with an immunosuppressive milieu. These differential roles are reflected by preferential recruitment of distinct subsets of T cells to site of inflammation. While CCL17 facilitates chemotaxis of effector T cells and supports DC-T cell interactions as well as DC migration towards CCR7-ligands, CCL22 induces chemotaxis of Treg cells. In addition, CCL22 signalling induces a more rapid desensitisation and internalisation of CCR4 than CCL17, suggesting biased agonism of CCL17 and CCL22. The functionality of CCL17 and CCL22 should, therefore, be considered in combination as well as individually in the context of immune-related diseases. The role of CCL17 and CCL22 in infectious diseases has not been well understood. The central hypothesis was that CCL17 and CCL22 play important but potentially different roles during bacterial infection. This was modelled using a well-studied bacterial pathogen, Salmonella enterica serovar Typhimurium (STM). It was hypothesised that CCL17 expression may direct the migration of STM-infected DC from the gut to draining lymph nodes a key bottleneck in early infection that controls bacterial dissemination to systemic sites. It was further hypothesised that CCL22 may play a role in immune regulation through the induction of Treg cells. These regulatory cells may have downstream effects on Th1 responses, which are critical for the control of Salmonella infection. In the first part of the thesis, the role of CCL17+ DC in the transmission of STM was investigated. Histological analysis of CCL17 reporter mice revealed that CCL17-expressing cells co-localised with Salmonella in the dome area of Peyer’s patches (PP). Further, CCL17-expressing DC contributed to dissemination of STM from PP to the mesenteric lymph nodes (mLN). Within the mLN, STM were found within CCL17+ DC as well as in other DC, monocytes and macrophages. Analysis of the STM+ DC subpopulations revealed that all DC subsets carried STM, but the CD103+ CD11b- DC could be identified as the main STM-containing population. STM infection triggered upregulation of CCL17 expression in specific intestinal DC subsets in a tissue-specific manner. Interestingly, the CD103+ DC subsets upregulated CCL17 in the PP, whereas CD103- DC subsets upregulated CCL17 in the mLN. In the second part of this thesis, the role of CCL17 and CCL22 in the induction of antigen-specific CD4+ T cell responses was investigated. CCL17/CCLL22 double-deficient, CCL17- and CCL22 single-deficient, and wild type mice were analysed after live-attenuated STM TAS2010 vaccination, vaccination/challenge and in steady-state. Mice deficient in both chemokines, CCL22 and CCL17, demonstrated a reduction of effector Treg cells. This promoted an enhanced STM-specific Th1 immune response characterised by an expansion of Th1 T cells, resulting in a more favourable effector Treg/activated Tconv ratio and a significantly improved vaccine efficacy to challenge with virulent Salmonella. In conclusion, the work presented within this thesis showed the contribution of CCL17+ DC in the dissemination of STM and identified CCL22 as a potential target to improve vaccine approaches.
Genomic approaches to stop the spread of bacterial infectious diseases
The advent of affordable whole genome sequencing has been a disruptive force that is changing the biological sciences, with public health microbiology being no exception. As microbial genomics is a new frontier for public health microbiology, a substantial amount of development and validation research is required. Genomic-based investigations in response to bacterial public health problems are gaining traction, but optimal approaches to bioinformatics and data analysis are still being developed. In order to help address these needs, this thesis investigated the utility of microbial genomics for Australian public health microbiology through three case studies of three distinct bacterial pathogens. Hospital adapted pathogens pose a significant health threat through their ability to rapidly emerge with adaptive phenotypes that permit them to circumvent infection control practices. In the first results chapter, comparative bacterial population genomics was used to characterise and better understand the genomic changes that have accompanied and driven the emergence of a novel and highly hospital associated lineage of Enterococcus faecium called ST796. These analyses revealed that the novel strain belonged to a highly hospital adapted lineage and shared a close evolutionary origin with another newly emerged hospital associated lineage of E. faecium. Complete assembly and analysis of a representative ST796 genome identified several key genomic events that were distinctive in the newly emerged lineage. Investigations of bacterial disease outbreaks caused by environmental pathogens have traditionally relied upon insights delivered through epidemiology supported by pathogen genotyping methods. Genotyping can be critical for identifying the source of an outbreak, but methods vary in their resolving power and genotyping data can be difficult to compare between investigations. In the second results chapter, a genomics-based machine learning approach was developed and used to predict the source of clinical Legionella pneumophila isolates using genomic patterns obtained prospectively through environmental sampling of bacteria. A proof-of-concept method was developed for assigning outbreak sources without using phylogenetic trees. Notably too, this study demonstrated phylogenetics can be misleading when assessing L. pneumophila environmental origins. The predictive statistical genomic method presented here has utility as a powerful tool for use alongside conventional L. pneumophila disease outbreak epidemiological investigations. Understanding a pathogen’s mode of transmission and infectious reservoir is a key goal in order to control the spread of disease, as such knowledge permits deployment of evidence-based strategies to manage outbreaks and prevent future occurrences. In the third results chapter, genomics was used to conduct high-resolution, micro-epidemiology population analyses on clinical isolates of another environmental bacterial pathogen, Mycobacterium ulcerans, the cause of Buruli ulcer. Bacterial population genomics and phylogeographic modeling suggested a westward migration of the pathogen across south east Australia that aligned well with the disease epidemiology. In addition, demographic models inferred from the genomic data provided a plausible explanation for a recent increase in cases of Buruli ulcer in south-eastern Australia. These insights demonstrate that M. ulcerans can be introduced to a new environment and then expands, rather than the awakening of a quiescent pathogen reservoir. Such discoveries inform our understanding of Buruli ulcer transmission and control. Collectively, these studies further demonstrate that microbial genomics significantly improves our understanding of the transmission dynamics behind clinically important pathogens over standard epidemiological investigations. This research demonstrates the advantages of the implementation of microbial genomics in public health microbiology.
The regulation of skin-homing receptors and its implications on T helper cells following localized Herpes Simplex Virus infection
Effective cellular immunity relies not only on the appropriate differentiation of antigen-specific T cells directed toward particular pathogens but also on the targeted migration of these cells to the sites of infection. E-selectin ligand (ESL) is a homing receptor widely recognized for its role in supporting the migration of leukocytes to the skin particularly during non-specific inflammation. However, the role of ESL in the migration of CD4+ T cells to the skin during viral infection and its regulation of expression on these cells are less understood. This thesis aimed to explore these aspects by utilizing an in vivo mouse model of localized skin infection with herpes simplex virus-1 (HSV-1) as well as a detailed investigation of in vitro polarized CD4+ T cells. Following epicutaneous but not intranasal HSV-1 infection, adoptively transferred transgenic HSV-specific CD4+ T cells induce the expression of ESL particularly in the infected skin. Similarly, genetic ablation of Fut7, a gene required for the generation of ESL, significantly decreased the capacity of CD4+ T cells to enter the skin of HSV-infected mice highlighting the importance of this homing receptor. While epicutaneous HSV infection of C57BL/6 mice predominantly induces a T helper type 1 (Th1) biased CD4+ T cell response, the data herein proved that the induction of ESL was not strictly related to Th1 differentiation of the responding HSV-specific CD4+ T cells. Interestingly CD4+ T cells deficient in the Th1 master regulator T-bet displayed a preference toward Th17 differentiation upon HSV-1 skin infection, yet still expressed high levels of ESL and there was no evidence of impaired migration to the infected skin. Further supporting this finding, in vitro analyses subsequently demonstrated the expression of ESL on both in vitro differentiated Th1 and Th17 cells but not Th2 cells. The glycosyltransferase core-2-beta-1,6-glucosaminyltransferase-I (C2GlcNAcT-I), which contributes to the synthesis of ESL epitope, appeared to be the primary determinant of the differential ESL expression between these Th subsets. As the differentiation of Th subsets and the induction of ESL in the draining lymph nodes in vivo both required TCR signaling and were evident prior to egress from the draining lymph nodes, the relationship between priming signals and ESL expression was assessed in vitro. The data indicated that both TCR signaling and the presence of exogenous cytokines were important for the induction and/or regulation of ESL expression. While TCR signaling was required for ESL expression, this signal alone was not sufficient. Rather the balance of cytokines, particularly of TGF-beta 1 and IL-4, had a marked impact on levels of ESL expression, with TGF-beta 1 acting to induce and IL-4 to suppress ESL expression on activated CD4+ T cells. Finally, this thesis investigated the temporal regulation of ESL induction during T cell priming and demonstrated that while the addition of TGF-beta 1 early during CD4+ T cell priming skewed the differentiation of Th subsets, the addition of this cytokine between 48-72 hours of T cell activation allowed for significant induction of ESL with only minimal interference on Th subset differentiation. Hence, the signals required for Th differentiation and ESL expression might actually occur in a consecutive manner to allow different Th subsets to express ESL and access the skin.
Characterisation of extracellular vesicles released upon NLRP3 activation and their impact on bystander cells
Inflammasomes are cytosolic multi-protein complexes that assemble upon detection of danger signals of infectious or sterile origin. Activation of the NOD-like receptor NLRP3 results in activation of caspase-1, which subsequently leads to cleavage and secretion of IL-1beta and IL-18, cytokines crucial for inflammation. In addition, NLRP3 activation has also been demonstrated to result in the secretion of extracellular vesicles (EVs). EVs are membraneous compartments, present in almost all body fluids, that derive either directly from the cell membrane (apoptotic bodies and ectosomes) or from multi-vesicular bodies (exosomes). They carry a set of proteins and RNAs distinct from their cell of origin and have been reported to be important mediators of cell-to-cell communication. Within this thesis I show that EVs can be isolated from conditioned tissue culture media most efficiently using a size exclusion chromatography-based protocol. This protocol enriches three distinct EV fractions: large EVs, intermediately sized EVs and small EVs. To investigate the impact of NLRP3 inflammasome activators on all EV subpopulations, THP-1 macrophages were treated with various NLRP3 inflammasome activators or different TLR ligands. All NLRP3 activators are capable of inducing EV secretion, which is primarily NLRP3- and caspase-1-dependent. TLR ligands also induce EV secretion, but in an NLRP3- and caspase-1-independent way. Comparisons of the RNA content of intermediately sized and small EVs show vast differences between the two EV subpopulations, suggesting a regulated loading of cargo into EVs. However, within the same EV subpopulation different NLRP3 activators cause the secretion of a very conserved RNA content, here defined as an EV-associated NLRP3 signature, which is divergent from the RNA content of TLR- induced EVs. Since activation of the NLRP3 inflammasome occurs in various inflammatory diseases, such as gout, atherosclerosis, type II diabetes and mutation-associated auto-inflammatory syndromes, this data may have clinical relevance for biomarker discovery. Additionally, transfer of NLRP3-induced EVs causes the induction of an interferon signature in EV recipient cells. Since interferons are known to counter-regulate inflammasome responses generally and I show here that NLRP3-induced EVs can dampen inflammasome responses in unprimed recipient cells specifically, the EV-induced interferon signature may provide an endogenous mechanism to prevent the induction of a systemic hyper-inflammatory state upon NLRP3 activation.
The role of T cell help in shaping Dendritic cell function
CD8+ T cell priming depends on antigen presentation by dendritic cells (DCs) and their capacity to communicate contextual cues associated with antigen acquisition. DCs often also require additional signals from helper CD4+ T cells, which upon mediation via CD40-CD40L further modulate the communication of contextual cue to the responding CD8+T cells. The present study was designed to explore the kinetics and molecular mechanisms underpinning this helper-dependent modulation of DC function. To address this, we employed an in vitro system of bone marrow (BM)-derived equivalents of CD8+ DCs (eCD8+ DCs) and we assessed the role of different CD40 signalling components in driving their IFN-aA-induced cytokine and chemokine responses by using flow cytometry, mass spectrometry-based proteomics, real time PCR and RNA sequencing. This brought to light remarkable and distinct patterns of gene regulation through which CD4+ T cells triggered CD40 and thereby amplified the capacity of IFN-aA to induce or downregulate a broad range of genes. We also observed an unexpected pattern of gene regulation: some genes required both T cell help and IFN-aA stimulations but could not be induced by ‘help’ or IFN-a alone. By varying the exposure time, we further discovered that eCD8+ DCs required 1-2 hours of IFN-aA to become responsive to CD40 triggering. Once this pre-activated state was achieved, CD40 stimulation rapidly amplified responses with remarkably fast kinetics. Combining proteomics and RNA sequencing data presented in this thesis suggests a complex interplay between the IFN-aA signalling pathway involving IRFs transcription factors and the NF-kB signalling pathway. These findings not only reveal new insights into how T cell help adjusts the responsiveness of DC to innate stimuli, but also reveal that this can occur with remarkable speed, which aligns with in vivo imaging studies describing very brief interactions between eCD8+ DCs and CD4+ T cells during CD8+ T cell priming.
Modelling melanoma control by immunotherapy and tissue-resident memory T cells using CRISPR/Cas9-based approaches
In recent years, immunotherapy has demonstrated remarkable efficacy in the treatment of metastatic melanoma due to the development of T cell-based therapies such as checkpoint inhibitors or adoptive T cell transfer therapy (ACT) directed against defined antigens. However, tumours frequently relapse from therapy by diverse acquired resistance mechanisms. Currently, it is not well understood how the choice of target antigen influences resistance mechanisms to antigen-specific immunotherapies. A better understanding of tumour recognition by the immune system is of utmost importance to further improve currently used immunotherapies. Therefore, we established CRISPR-assisted insertion of epitopes (CRISPitope), a technique that fuses a model CD8+ T cell epitope, human gp100, to endogenous gene products. We applied CRISPitope to murine melanoma cells to tag the endogenous melanosomal protein, TYRP1, and the oncogenic protein, CDK4R24C, with the same model epitope, rendering them targetable by the same TCR-transgenic T cells. This defined experimental setting enabled us to investigate how the choice of the targeted gene product impacts on therapy outcome and immune evasion mechanisms. Using experimental mouse models, we could identify different escape mechanisms to gp100-specific immunotherapy in TYRP1 versus CDK4R24C melanomas. Resistance to ACT targeting TYRP1 was mainly caused by permanent antigen loss, accompanied by a non-inflamed microenvironment, or reversible downregulation of the antigen associated with melanoma phenotype switching. In contrast, CDK4R24C melanomas escaping ACT displayed antigen persistence and were associated with an IFN-rich inflamed tumour microenvironment. In CDK4R24C melanomas IFN-driven feedback inhibition by negative immune-checkpoint molecules promoted resistance to ACT despite persistent antigen expression. Applying CRISPitope to syngeneic mouse models, we could show that target antigen choice can influence ACT resistance mechanisms, phenotype and immune contexture of melanomas in response to antigen-specific immunotherapies. Thus, our work could help to better understand acquired resistance and optimise personalised cancer immunotherapy. Furthermore, we aimed to apply this platform to a model of melanoma immune surveillance by TRM cells in order to understand the importance of cognate antigen expression and presentation for long-term tumour control by CD8+ tissue-resident memory T cells (TRM). To address this question, we used a modified CRISPitope-approach, called SWITCHitope, to generate melanoma cell lines that express a floxed model antigen under the control of an endogenous promoter and a Tamoxifen-inducible Cre-recombinase. We could confirm successful Tamoxifen-inducible depletion of the model antigen in melanoma cells in vitro and in vivo. Moreover, we showed that antigen-depleted melanoma cells have significantly reduced potential to activate TCR-transgenic T cells in vitro. Using a transplantable epicutaneous melanoma inoculation technique, we could demonstrate that SWITCHitope-engineered melanoma cells can prime naïve T cells, recruit them into the skin and induce T cell differentiation towards a TRM phenotype. Our approach enables us to investigate the importance of antigen expression and presentation for TRM melanoma control. This work will help to better understand the interplay between tumour cells and TRM cells and thereby advance clinical translation.
Essential and complementary function of dendritic cell subsets during immunotherapy
T lymphocytes are critical components of the adaptive immune system that protects us against a variety of infections. However, during chronic infections such as with HIV, HCV and HBV as well as cancers, T cells become functionally impaired in order to limit immunopathology. This in turn allows these infections to persist or tumours to grow and spread. Scientific research over the last decades lead to the development of checkpoint inhibitors which can reinvigorate exhausted CD8+ T cells. This enables them to fight persisting infections and to eliminate even advanced tumours. Based on this success, checkpoint immunotherapy has revolutionized cancer treatment. With the identification of the memory-like TCF-1+ subset of exhausted CD8+ T cells that responds to checkpoint immunotherapy, predictions for clinical responses can be further improved. Yet, despite the enormous success and routine application, a deeper mechanistic understanding of checkpoint immunotherapy is required in order to help patients in whom this therapy failed. In this study, we elucidate critical cellular interaction partners of exhausted CD8+ T cells during anti-PD-L1 treatment. Using the murine chronic LCMV model, we found that DC with their key function in T cell activation are pivotal for a successful therapy demonstrated by the expansion of virus-specific CD8+ T cells and control of viral load. Notably, different DC subsets represent complementary roles in this context. The cross-presenting subset of XCR1+ DC are critical to maintain the population of memory-like TCF-1+ exhausted CD8+ T cells while the remaining DC promote proliferation of such. Data presented in this study indicate that a complex network of signalling molecules delivered by DC subsets is involved in this process. Performing detailed transcriptional analysis of memory-like TCF-1+ cells, we deciphered that this cell pool represents a heterogenous population and our results imply that the different subsets reside in different areas of secondary lymphoid organs. Investigation of the microanatomy of the CD8+ T cell – DC interaction revealed three distinct areas of the spleen where communication of the cellular subsets occurs. Taken together, we elucidated a complex interplay between exhausted CD8+ T cells and DC during chronic viral infection and anti-PD-L1 treatment. This communication impacts on the transcriptional and functional level of exhausted CD8+ T cells as well as on their localization in secondary lymphoid organs. Overall, our data identified critical cellular interaction partners and their specific anatomical localization that is essential for the successful reinvigoration of exhausted CD8+ T cells during checkpoint immunotherapy.
The role of cytohesins in the regulation of immune responses
Cytohesins are guanine nucleotide exchange factors for adenosine diphosphate ribosylation factor (Arf) proteins and promote the switch of Arfs to the active GTP-bound form. Cytohesins have been shown in different in vitro settings to affect cell motility, cell adhesion and chemotaxis of various leukocytes, which are fundamental processes necessary for efficient innate and adaptive immune responses. Furthermore, due to their engagement in phagocytic processes, cytohesins are also targeted by different pathogens during bacterial invasion to evade the immune responses and to exert their full pathogenicity. However, all the evidence for the regulation of immunity by cytohesins has derived from in vitro studies. The primary impact(s) of different cytohesins on the regulation and coordination of the immune responses in the control of infection in vivo has not been elucidated. The aim of this PhD thesis was to investigate the in vivo function of cytohesin-1, cytohesin-2 and cytohesin-3 in the complex immune responses and in pathogenesis by using acute infection with the respiratory pathogens Legionella pneumophila and influenza A virus in knockout mice. L. pneumophila is a Gram-negative bacteria and the causative agent for Legionnaires’ Disease, and influenza A virus causes 'flu', which occurs in seasonal and pandemic outbreaks. These studies revealed that cytohesin-1 promotes T cell responses in both bacterial and viral respiratory infections. Moreover, in influenza A infection, cytohesin-1 deficiency hampered development of cognate T cells and their response to cognate antigens. Cytohesin-1 was demonstrated experimentally to be involved in the initial activation phase of naive T cells and was required for optimal metabolic switching of T cells following activation. Lack of cytohesin-1 impaired the differentiation of distinct helper T cells, but also different memory and effector cell types. Myeloid-specific deletion of cytohesin-2 transiently impaired cDC recruitment in the course of bacterial infection highlighting a potential intrinsic role in cDC biology. However, this did not have major effects on the overall phenotype of L. pneumophila or influenza A infection. Interestingly, cytohesin-3 had an opposing role on T cells compared to cytohesin-1 and suppressed T cell immune responses in both L. pneumophila and influenza A infection. Increased infiltration of several different T cell subpopulations to the site of infection and increased acquisition of antigen-specific responses was observed in cytohesin-3 deficient mice. Furthermore, cytohesin-3 deficient T cells were more reactive to cognate stimulation leading to enhanced cellular immune responses. Additionally, recovery from L. pneumophila infection was delayed in cytohesin-3 deficient mice, suggesting that cytohesin-3 is important for preventing overactivation of T cells and any resulting inflammatory disease. In conclusion, this PhD thesis provided for the first time a broad in vivo examination of the role(s) of different cytohesins in the immune responses to pulmonary infections. Although minor roles were found for cytohesins in regulating innate immune responses, the primary role(s) of cytohesin-1 and cytohesin-3 appear to lie in the regulation of T cells. Cytohesin-1 promotes T cell responses potentially by providing the optimal (signalling) threshold and by supporting the bioenergetic adaptation following T cell activation, while cytohesin-3 may suppress T cell responses by acting as an immune checkpoint.
Impact of chronic liver inflammation on adaptive immune responses to viral infection
A common complication in patients suffering from chronic liver inflammation and fibrosis is the enhanced susceptibility to viral infections and weak responses to vaccination, which are associated with significant co-morbidities. To unravel the cellular and molecular mechanisms underlying the impaired adaptive immune response during liver fibrosis, we investigated the T cell-mediated immune responses to lymphocytic choriomenigitis virus (LCMV) infection, as well as in response to OVA/PolyI:C vaccination. Using the bile duct ligation (BDL) murine model of chronic liver inflammation and fibrosis, we found that chronic liver damage is associated with persistence of infection, recapitulating the clinical situation in humans. Hallmarks of the defective anti-viral immunity in these mice were reduced expansion of LCMV-specific CD4+ and CD8+ T cells, decreased expression of IFNg and TNFa, as well as an elevated co-expression of the exhaustion marker PD1 together with LAG3 and TIM3 in virus-specific T cells from the spleen and liver. The reduced number of LCMV-specific CD4+ and CD8+ T cells are due to decreased proliferation as well as increased apoptosis among SMARTA T cells. Additionally, LCMV-specific CD4+ and CD8+ T cells display reduced mitochondrial fitness, characterized by a higher proportion of cells containing depolarized (i.e. dysfunctional) mitochondria, and producing high levels of superoxide among SMARTA and P14 T cells from mice with liver fibrosis. After OVA/PolyI:C vaccination antigen-specific CD4+ and CD8+ T cells were similarly reduced in numbers in mice with liver fibrosis and displayed reduced production of IFNg, TNFa and IL-2. Interestingly, endogenous OVA-specific CD8+ T cells, as well as transferred OT-I T cells also show severe signs of exhaustion after vaccination, which manifested in elevated levels and co-expression of PD-1 and LAG3, as well as PD-1 and TIM3 in mice with liver fibrosis. The future goal of this project is to identify key molecular pathways induced by chronic liver damage that can be therapeutically modulated to promote anti-viral immunity and to improve vaccination responses in patients suffering from chronic liver inflammation and fibrosis.
Function and diversity of myeloid cells during chronic inflammation in non-lymphoid organs
Different immune cells play a major role in maintaining tissue homeostasis, tissue repair but also in inflammation. Tissue integrity is mainly controlled by tissue regulatory T cells (Tregs), type 2 innate lymphoid cells (ILC2) and myeloid cells. Here, we are using a chronic kidney disease (CKD) model of adenine enriched diet induced crystal-nephropathy and high-fat diet induced obesity to investigate the contribution of Tregs, macrophages and dendritic cells during inflammation. Using a modified gating strategy to distinguish macrophages and conventional dendritic cells (cDCs) we detected that macrophages and cDCs infiltrate the visceral adipose tissue (VAT) during obesity. Here, sex-specific differences show that macrophages and cDCs accumulate significantly more in male VAT. RNA sequencing confirmed a higher pro-inflammatory gene signature in male VAT macrophages and cDCs. Those data indicate that myeloid cells might be a significant contributor to the sex-specific differences in the adipose tissue. Interleukin (IL)-33 maintaining tissue homeostasis by promoting Tregs and ILC2s in the VAT. Investigating IL-33 and its receptor ST2 during kidney inflammation in the adenine enriched diet induced crystal-nephropathy we show that ST2 deficiency is negligible in a severe CKD onset, whereas using a mild disease onset we detect a disease exacerbation. Additionally, we show that the deficiency for IL-33 leads to a more severe disease progression accompanied by a high influx of several immune cells and lower frequency of effector Tregs. Transcription factors such as IRF1, IRF4 and BATF are linked to regulate suppressive cell functions e.g. in Tregs and macrophages. Using transcription factor knockout mice, we determined that IRF1 deficiency leads to disease amelioration by reducing macrophage and monocyte infiltration. IRF4 and BATF deficiency lead to reduced numbers of Tregs in the kidney. IRF4-/- but not BATF-/- showed significant increased myeloid cell infiltration. Bone marrow chimera reconstituted with IRF4 deficient CD4 T cells show reduced numbers of Tregs in the inflamed kidney and correlated with the myeloid cell infiltration. Collectively, the findings point to a sex-specific contribution of macrophages and cDCs in tissue inflammation and indicate that the IL-33/ST2 axis regulates immunosuppressive cells tissue and disease progression specific.