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- Grüttner, Jana, 1992-
(author)
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The interplay between Giardia intestinalis and host intestinal epithelial cells : A tale of immune activation and suppression
- 2023
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Doctoral thesis (other academic/artistic)abstract
- Giardia intestinalis is a non-invasive, protozoan parasite causing the diarrheal disease giardiasis in a variety of mammals, including humans. The parasite has two main life cycle stages; disease-causing trophozoites and infectious cysts. Trophozoites colonize the host’s upper small intestine by attaching to intestinal epithelial cells (IECs). G. intestinalis infections can cause a broad spectrum of clinical outcomes, ranging from acute and chronic infections to asymptomatic carriage. In most cases, the infection causes only a low degree of mucosal inflammation. The molecular basis of how the parasite causes such diverse clinical outcomes is poorly defined. Here, we used in vitro infection models to investigate host-parasite interactions between G. intestinalis and IECs. In paper I, we assessed transcriptional changes of Caco-2 cells during infections with different life cycle stages of the parasite by RNA-sequencing (RNA-seq). Infections with trophozoites, cysts or encysting cells showed that each life cycle stage of the parasite induced the expression of both a common set of host genes and unique, life cycle stage specific responses. The IECs showed large expression changes in immune signalling, transcriptional regulation, apoptosis, metabolism and oxidative stress. We could also observe that the parasites’ contact with IECs inhibited its encystation processes. In paper II, we mapped global protein changes of Caco-2 cells during G. intestinalis trophozoite infections using quantitative proteomics. We could confirm expression changes in oxidative stress, immune response, signalling, metabolism and apoptosis on protein level. We further investigated the interplay between IECs and G. intestinalis using small intestinal organoids, to more accurately mimic intact primary host tissue. The infection dynamics of trophozoites on intestinal organoid-derived monolayers were assessed using live-cell microscopy (paper III). We investigated swimming behaviour of trophozoites on the intestinal epithelial surface. Trophozoites swan in repetitive circling and intermittent attachment patterns during preattachment swimming, possibly to explore the epithelium surface and to find a suitable permanent attachment site. We further assessed early immune responses of IECs during trophozoite infections using intestinal organoid-derived monolayers (paper IV). Transcriptional expression of immune response genes was dependent on the trophozoite inoculate. Trophozoites that were preconditioned to maximize the trophozoites’ fitness triggered only a negligible inflammatory transcriptional response, while lysed and “non-fit” trophozoites induced a strong inflammatory response. “Fit” trophozoites could even suppress the inflammatory response triggered by lysed trophozoites, demonstrating the immune modulating capacity of the parasite. In conclusion, this thesis has increased our understanding of the interplay between G. intestinalis and IECs.
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- Tadala, Lalitha, 1991-
(author)
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Role of extracellular ATP in immune mechanisms against infections
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Inflammation is driven either by infection with pathogens or sterile stimuli, such as tissue damage or autoimmune diseases. Upon tissue damage, ATP is released passively from the dead or compromised cells. During stress, ATP can be secreted from the cells. Extracellular ATP (eATP) acts as an endogenous danger signal. An increase in eATP is sensed by cell surface purinergic receptors and regulates the onset and resolution of inflammation. Extracellular ATP is an important inflammatory mediator during sterile inflammation. On the other hand, the role of eATP is poorly studied during infection, both bacterial and viral. In this thesis, I present the molecular mechanisms underlying ATP secretion during bacterial infections and the role of eATP in human hantaviral infections.During infection with certain enteropathogenic Gram-negative bacteria, intestinal epithelial cells secrete ATP via connexin hemichannels as an alert signal to activate the immune system, which triggers acute inflammation in the gut. However, neither what triggers ATP secretion nor the molecular mechanisms of ATP secretion were known. Pharmacological, genetic, and microscopy-based evidence shows that during invasive bacterial infections, the plasma membrane ruffles act as mechanical immune stimuli and activate the inherently mechanosensitive plasma membrane channel PIEZO1. Mechanically activated PIEZO1 leads to the influx of Ca2+ ions and concurrent ATP secretion. In addition, PIEZO1 also activates protective transcriptional responses. Thus, PIEZO1 acts as a sensor for invasive infection using mechanical stimuli, unlike the so-far-described immune sensors of infection, which all recognize microbial components by chemical interaction.During human hantavirus infection, the humoral immune responses are poorly studied. Our collaborators found that atypical B cells, which do not have the surface marker CD27, show increased frequency in a cohort of hantavirus-infected patients. CD27 shedding in murine lymphocytes had been previously linked to eATP-dependent activation of a purinergic receptor7. To test whether ATP levels in the circulation of hantavirus-infected patients are elevated, an approach to perform same-day eATP quantifications in human plasma was developed. This assay was used to establish the normal eATP concentration in plasma in a cohort of healthy volunteers and to show that eATP levels are elevated in the acute and convalescent stages of hantavirus infection. Further, the addition of ATP to isolated human B cells recapitulated the observed CD27 shedding via a metallomatrix proteinase-8-dependent (MMP8) mechanism. Together, these projects provide evidence for the importance of eATP in bacterial and viral infectious diseases.
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