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Item Open Access Efficient Methods for Ex Vivo Generation of Gene-edited NeutrophilsNg, AnthonyNeutrophils are the most numerous circulating leukocyte in humans and are key first responders to invading pathogens and effectors of tissue repair and homeostasis. The potent antimicrobial functions of mature neutrophils are tightly controlled due to their potential for tissue damage, and thus neutrophil dysregulation is a central feature in many inflammatory and infectious diseases. Mature human neutrophils are challenging to study due to their short lifespan in culture and propensity to activate to diverse stimuli making the genetic modification of mature human neutrophils impossible, an important technical limitation in the field of neutrophil biology. Here I demonstrate that primary human haematopoietic progenitors isolated from the circulation of healthy adults can be expanded and directed to differentiate into mature neutrophils using granulocyte colony stimulating factor, and that these cells recapitulate multiple functions of mature circulating neutrophils. Stable modification of progenitors by Cas9 ribonucleoproteins or lentiviral vectors followed by granulocytic differentiation yields uniform populations of gene edited neutrophils in an activation-free manner, allowing the interrogation of individual genes in the neutrophil context. I use lentiviral vectors to validate the pro-efferocytic function of neutrophil TIMD4 and show its trafficking to membranes during apoptosis, uncovering a novel mechanism by which apoptotic neutrophils actively regulate their clearance by phagocytes. I then show that Cas9 ribonucleoprotein delivery by nucleofection is capable of stable knockout of the highly expressed gene *ITGAM* and that loss of its product CD11b, a key component of the complement receptor CR3, does not impair neutrophil phagocytosis of serum opsonised bacterial bioparticles, contrary to current evidence. This method for the efficient, flexible, and scalable generation of gene-edited primary neutrophils represents a powerful tool capable of discovering truly novel facets of neutrophil biology, the understanding of which is critical in the rational design of therapeutic strategies in a diverse array of infectious and inflammatory diseases.Item Embargo N-Glycosylation Protects Host from Mycobacterial InfectionLyu, BingnanHost immunity against tuberculosis (TB) is primarily mediated by macrophages. Macrophages phagocytose *Mycobacterium tuberculosis* and restrict their growth. Through a zebrafish forward genetic screen, we have identified the early steps of N-glycosylation are required for the macrophage to resist mycobacterial infections. N-glycosylation attaches an oligosaccharide molecule to nascent secretory or membrane proteins within the endoplasmic reticulum (ER) for protein folding and trafficking. Defects of N-glycosylation induce the unfolded protein response (UPR), which sensitizes infected macrophages to intrinsic apoptosis. This apoptosis differs from the cell death observed in infected wild-type cells and is not dependent on mycobacterial virulence factors. Therefore, N-glycosylation provides protection by mitigating the ER stress of protein synthesis triggered by cellular responses to mycobacterial infection.Item Embargo A genomic investigation into gastrointestinal antimicrobial resistance and clinical infection in patients undergoing transplantationMadhav, ArchanaGlobally, antimicrobial-resistant bloodstream infections (AMR-BSIs) claim 1.5 million lives annually. High-resolution sequencing data allows us to derive novel insights into AMR-BSIs. Immunosuppressed patients have a disrupted gut microbiome; therefore, transplant recipients provide an ideal reservoir for populations of opportunistic AMR pathogens to flourish. Specifically, clinical infections caused by enteric AMR bacteria that translocate across the intestinal epithelium pose a significant barrier to the improvement of transplant outcomes worldwide. My thesis re-examines the significant body of research that encompasses gut-origin sepsis and contextualises it within the ongoing AMR crisis. I combined whole-genome and metagenomic sequencing approaches to 1) characterise BSI-associated AMR; 2) study the phenomenon of bacterial translocation in two distinct cohorts of transplant recipients – Small Bowel Transplantation and Haematopoietic Stem Cell Transplantation; and 3) illustrate the longitudinal evolution of AMR in these transplant cohorts. Using genomic data, I developed high-throughput screening strategies to aid in the monitoring and mitigation of post-transplant infectious complications caused by gut-residing pathogens. Enteric *E. coli*, *Klebsiella* spp. and *Enterococcus* spp. were identified as significant contributors to the cumulative burdens of BSI and AMR. These organisms also showed evidence of translocation and temporally diversified transplant recipients’ gut resistomes. Investigating these resistomes revealed patterns in paired AMR gene co-localisation, suggesting links with mobile genetic elements and potential for transmission via horizontal gene transfer (HGT). In summary, improved surveillance of HGT can bolster efforts to ensure judicious use of existing antimicrobials and optimise treatment regimens to minimise inadvertent selection for resistance among patients undergoing transplantation.Item Open Access High-throughput sequencing to understand pathogen carriage and transmissionAggarwal, Dinesh; Aggarwal, Dinesh [0000-0002-5938-8172]Genomic sequencing is a powerful technology in the study of pathogen biology and pathogen transmission. In this thesis I have studied the human nasal carriage of *Staphylococcus aureus* and, in response to the SARS-CoV-2 pandemic, I examine the transmission of SARS-CoV-2, two critically important human pathogens. To address this, I have utilised genomic and individual-level epidemiological data, acquired through the CARRIAGE study and the COVID-19 Genomics UK Consortium (COG-UK). I characterised the microbial community structure that facilitates or prevents persistent nasal colonisation by *Staphylococcus aureus* using 16S rRNA gene sequencing of serially collected nasal swabs from 1,180 individuals. I provide evidence, by defining the carriage status of participants, that alpha and beta diversity of the anterior nares significantly differs by carriage status. I demonstrate that persistent carriage in the anterior nares is negatively associated with multiple species, compared with other carriage states. These findings redefine intermittent carriage, which does not appear to be defined by a distinct microbial community. Using a random forest model, I show that *S. aureus* carriage can be predicted from microbiome data with a moderate degree of accuracy, and explore the phylogenetic relationship of *S. aureus* isolates recovered from nasal swabs with their associated microbiome. To facilitate higher throughput sequencing in the CARRIAGE study, I optimized 16S rRNA gene sequencing. Using nasal samples from healthy human participants and a serially diluted mock microbial community, I compared alpha and beta diversity, and compositional abundance where the PCR amplification was conducted in triplicate, duplicate or as a single reaction, and where manually prepared or premixed mastermix was used. I find no requirement for pooling of PCR amplifications or manual preparation of PCR mastermix, resulting in a more efficient 16S rRNA gene PCR protocol. Moreover, I demonstrate the need for sufficient controls to account for contaminants, which are an important source of bias. I used care homes as a model to understand the utility of whole genome sequencing early in the SARS-CoV-2 pandemic. I reviewed all genomic epidemiology studies on SARS-CoV-2 in care-homes that had been published up to 3 November 2020 and identified numerous sources of, and opportunities to mitigate, transmission of SARS-CoV-2 in care homes using genomics. Importantly, I recognised the importance of combining detailed epidemiological data with SARS-CoV-2 genomic data to inform public health decision making and detail sources of possible error introduced through bioinformatic analysis in multiple studies. To identify the transmission dynamics of SARS-CoV-2 in a university population, I used 482 prospectively sequenced SARS-CoV-2 isolates derived from asymptomatic student screening and symptomatic testing of students and staff at the University of Cambridge from 5 October to 6 December 2020. I performed a detailed phylogenetic comparison with 972 isolates from the surrounding community, complemented with epidemiological and contact tracing data, to determine transmission dynamics. I found that after a limited number of viral introductions into the university, the majority of student cases were linked to a single genetic cluster, likely dispersed across the university following social gatherings at a venue outside the university. I identified considerable onward transmission associated with student accommodation and courses; this was effectively contained using local infection control measures and dramatically reduced following a national lockdown. I observed that transmission clusters were largely segregated within the university or within the community. To characterise the contribution of imported SARS-CoV-2 to onwards transmission and establishment of SARS-CoV-2, I evaluated the effectiveness of travellers being required to quarantine for 14 days on return to England in Summer 2020. I identified 4,207 travel-related SARS-CoV-2 cases and their 18,885 contacts, along with 888 associated travel-related SARS-CoV-2 genomes in the UK SARS-CoV-2 sequencing dataset. Quarantining was associated with a lower rate of contacts. Fewer genomically-linked cases were observed for index cases who returned from countries with quarantine requirement compared to cases from countries with no quarantine requirement, but this effect was explained when adjusting for the number of importations for each genome. Furthermore, I identified a large travel-related cluster dispersed across England, which was confirmed with contact-tracing data. In conclusion, I have described the microbial basis for *Staphylococcus aureus* colonisation of the anterior nares; importantly I have re-defined carriage and developed an effective model for predicting carriage status. I have identified the key determinants of SARS-CoV-2 transmission and effective interventions in care homes, a large UK university, and at a national scale, highlighting the clear utility of whole-genome sequencing to inform real-time public health policy.Item Open Access The Role of BMP9-Induced SEMA3G in Pulmonary Vascular StabilityMirza, SarahThe progressive disease, pulmonary arterial hypertension (PAH), occurs when the pulmonary vasculature pathologically remodels and constricts, causing increased pulmonary arterial pressure eventually leading to right heart failure. Loss of function mutations in the bone morphogenetic protein (BMP) pathway are known to be causative for PAH. Mutations in the type II receptor BMPR-II are the main cause of familial PAH and are also found in sporadic idiopathic PAH patients. BMP9 therapy is being investigated in PAH, as it increases *BMPR2* expression, inhibits endothelial cell permeability, and reverses vessel remodeling in PAH preclinical models. The exact mechanism by which BMP9 regulates vessel stability is unknown, but BMP9 is known to inhibit pro-angiogenic VEGF signalling. A microarray study conducted by the Morrell group in pulmonary artery endothelial cells after treatment with BMP9 revealed Semaphorin 3G (SEMA3G) as a potential BMP9-regulated gene. Class 3 semaphorins have been implicated in angiogenesis and endothelial cell migration, with several family members, including SEMA3G, inhibiting VEGF signalling via competition for the neuropilin co-receptors. Furthermore, SEMA3G was shown to prevent pathological vascular remodelling in diabetic and oxygen-induced retinopathies. These findings suggest that SEMA3G could play an important role in maintaining the stability and integrity of the vasculature. The work presented in this thesis confirms that BMP9 and BMP10 treatment of pulmonary microvascular endothelial cells (PMECs) increases *SEMA3G* expression 5-10-fold. Knockdown of BMP signalling pathway components revealed that BMP9/10 regulated *SEMA3G* expression via the canonical Smad signalling pathway. Involvement of SOX17 in *SEMA3G* upregulation was also observed. Additionally, *Sema3g* expression was reduced in *Bmp9* knockout mice and in an experimental animal model of PAH. Interestingly, VEGF treatment inhibited the BMP9-mediated increase of *SEMA3G* expression. Conversely, I found that SEMA3G could inhibit VEGF-mediated phosphorylation of VEGFR2. *In vitro* endothelial cell assays were used to examine the functional consequence of BMP9-mediated *SEMA3G* expression. This work confirmed that BMP9 inhibits VEGF-induced endothelial cell migration and tube network formation in PMECs, and subsequently demonstrated that *SEMA3G* is essential for BMP9-mediated inhibition of migration and tube network formation. Therefore, the work presented here suggests that SEMA3G plays an important role in balancing pro-angiogenic VEGF and anti-angiogenic BMP9 signalling. This study further elucidates the mechanism by which BMP9 functions as a vascular quiescence factor in healthy vasculature and elucidates mechanisms by which BMP9 might act therapeutically in PAH.Item Embargo Investigating the role of the UDP-galactose transporter SLC35A2 in the regulation of HIF signallingGreef, BasmaBackground:
The hypoxia inducible factor (HIF) family of dimeric transcription factors play a vital role in the cellular response to low oxygen (hypoxia). HIF activation leads to the transcriptional upregulation of a range of genes that are involved in diverse physiological and pathophysiological processes. In malignant tumours increased HIF-α expression, and HIF activation are commonly observed, and are associated with poorer prognosis and disease progression. HIF activity is controlled by a number of different mechanisms, and the identification of molecular regulators of HIF is of intense interest. The Golgi UDP-galactose transporter SLC35A2 was identified by the Ashcroft group as a novel regulator of HIF-α, and CHO cells that had lost SLC35A2 exhibited elevated HIF-α protein in normoxia. It is known that the absence of SLC35A2 in the Golgi leads to wide-ranging glycosylation defects. The underlying mechanisms linking SLC35A2 to HIF-α protein regulation were not known. This thesis aims to further characterise the role effect of SLC35A2 loss on HIF signalling and tumour cell behaviour, in CHO cells and a human cell system, specifically by exploring the hypotheses that these cells may harbour an autophagy defect, or differences in overall UDP-sugar content, in particular O-GlcNAc. Methods and Results: I used parental and Slc35a2 mutant CHO cells, SLC35A2 knockout (KO) and wild type (WT) suspension HeLa (sHeLa) cells, and a panel of cancer cells lines, including 786O, RCC4 renal carcinoma cells which exhibit constitutive HIF activation due to loss of VHL function. Confirming previous work from the Ashcroft group, I showed that Slc35a2 mutant (M6.19) CHO cells exhibit elevated normoxic HIF-1α protein levels and evidence of a glycosylation defect. Expanding on these findings, I found that SLC35A2 loss led to elevated normoxic HIF-α (HIF-1α and HIF-2α) protein and *HIFA* mRNΑ, and elevated expression of HIF-α target genes (*GLUT1*, *VEGF*). Using global RNAseq analysis of SLC35A2 KO and WT sHeLa cells, I found that SLC35A2 loss was associated with highly upregulated expression of a range of genes. Further exploration of the observed glycosylation defect in Slc35a2 mutant CHO cells, showed that SLC35A2 KO sHeLa cells also exhibited altered mobility of GLUT1 protein, which I found was consistent with treatment of WT sHeLa cells with tunicamycin, a protein *N*-glycosylation inhibitor. UDP-sugar analysis by HPLC of Slc35a2 mutant CHO and SLC35A2 KO sHeLa cells indicated increased levels of UDP-GlcNAc compared to their WT counterparts. Furthermore, I found similar patterns of increased UDP-GlcNAc levels in SLC35A2 KO compared to WT sHeLa cells in normoxia and hypoxia, while stable reconstitution of Slc35a2 in Slc35a2 mutant (M6.19) CHO cells reduced levels of UDP-GlcNAc and rescued CMP-sialic acid levels. Consistent with my findings and the involvement of UDP-GlcNAc in glutamine metabolism, I found that Slc35a2 mutant (M6.19) CHO cells were significantly more sensitive to glutamine withdrawal compared to parental (C4.5) CHO cells. Slc35a2 mutant (M6.19) and SLC35A2 KO sHeLa cells exhibited a glycosylation defect of LAMP2A, a protein involved in HIF-1α lysosomal degradation and critical for chaperone mediated autophagy. SLC35A2 mutant (M6.19) CHO cells were significantly more sensitive to the inhibition of proliferation by the inhibitors of autophagy bafilomycin and 3-methyladenine, and showed elevated levels of the autophagy marker LC3B. Finally, from analyses of the TCGA-KIRC database I found *SCL35A2* expression was higher in patients with non-mutant VHL versus mutant VHL in renal cancers. In support of these findings, I showed that basal SLC35A2 protein and mRNA levels were higher in patient-derived 786O renal carcinoma cells reconstituted with wild type VHL (786O-VHL) compared to matched 786-O empty vector control (786O-EV) cells. I also found that high *SLC35A2* expression is associated with poor prognosis in patients with *VHL* non-mutant ccRCC. Conclusion: Taken together, my thesis identifies SLC35A2 as a regulator of HIF-α and metabolism, potentially through its role in regulating UDP-sugars, and reveals a possible novel role for SLC35A2 in lysosomal processing and autophagy. *SLC35A2* expression in renal cell carcinoma is associated with VHL status, which may provide a new route for dysregulation of HIF, altered metabolism, and changes in lysosomal processing and autophagy.Item Embargo Imaging Post-Infarct Myocardial Inflammation with ⁶⁸Ga-DOTATATE PET/MRI and insights into COVID-19 associated myocardial injuryCorovic, Andreji) Post-Infarct Myocardial Inflammation and Imaging Background: Inflammation and its resolution modulate post-infarct myocardial injury. An excessive or prolonged inflammatory phase after myocardial infarction (MI) may contribute to adverse cardiac remodelling. 68Ga-DOTATATE is a PET radiotracer with high affinity for somatostatin receptor subtype 2 (SST2), which has been shown to be up-regulated in pro-inflammatory macrophages. Aims: To investigate the role of hybrid 68Ga-DOTATATE PET/MRI for tracking post-infarct myocardial inflammation and predicting adverse cardiac remodelling post-MI, as well as to identify potential systemic and blood biomarker correlates of myocardial 68Ga-DOTATATE PET signal. Methods: In a prospective observational cohort study, participants with recent MI underwent hybrid 68Ga-DOTATATE PET/MRI within 2 weeks of infarction (t0), and again after 3 months (t3M). Participants additionally underwent CT coronary angiography at baseline, and a full cardiac MRI scan at 1 year (t1Y). Participants with a prior history of MI, heart failure, previous coronary intervention or bypass grafting, were excluded. Blood samples were taken at the time of imaging for the quantification of circulating immune cell subsets using mass cytometry by time-of-flight (CyTOF) and for serum proteomic analysis. 68Ga-DOTATATE maximum Standardised Uptake Values (SUVmax) were derived for myocardial segments with late gadolinium enhancement (LGE; “infarct”) as well as for segments without LGE and myocardium remote from the infarct. Assessments of regional wall motion; segmental strain and T1/T2 mapping values; left ventricular volumes and biplane ejection fraction, were performed from cardiac MRI at t0 and t1Y. Regions of interest for quantification of 68Ga-DOTATATE PET signal were also derived for the ascending and descending aorta, vertebrae and non-culprit coronary arteries. *Ex vivo*, cardiac histological specimens from patients with a recent history of MI or ischaemic cardiomyopathy were immuno-stained for SST2, CD3, CD80, CD206/mannose receptor and CD68. Results: 38 participants in total were recruited (mean age 60 [SD 9] years; 32 [84%] male and 6 [16%] female), of whom 22 (58%) had ST elevation MI and 16 (42%) had non-ST elevation MI. The mean peak troponin at baseline was 16665ng/L (range 408 to >25,000ng/L) and the mean LVEF on pre-discharge echocardiogram was 49% (range 28%-61%). 36 (95%) participants had a follow-up 68Ga-DOTATATE PET/MRI at t3M and all 38 participants had a full cardiac MRI at t1Y. At t0, 68Ga-DOTATATE PET signal demonstrated a clear ability to distinguish infarct from remote myocardial regions. Furthermore, on segmental analysis, akinetic or hypo-kinetic myocardial segments has a significantly higher mean SUVmax than normo-kinetic segments. Segmental myocardial 68Ga-DOTATATE SUV also significantly correlated with peak segmental strain and segmental T1 values. At t3M, there was a significant decline in infarct 68Ga-DOTATATE SUV, in keeping with resolving myocardial inflammation, which was paralleled by a decline in high sensitivity Troponin, high sensitivity C reactive protein and NTproBNP levels. Immunostaining of histological specimens from patients with recent MI or ischaemic cardiomyopathy revealed co-localisation of SST2 with CD68, supporting the conclusion that the myocardial 68Ga-DOTATATE PET signal was derived, at least in part, from macrophages. After multivariable adjustment, the mean SUVmax of the infarct at t3M, but not t0, was significantly positively associated with left ventricular dilatation at t1Y (delta EDVi; p=0.038). The t3M/t0 ratio of the mean infarct SUVmax appeared to be an even stronger predictor of volumetric remodelling outcome. After statistical feature selection and multiple comparisons adjustments, a number of biomarkers were associated with the post-MI myocardial PET signal, including serum programmed death-ligand 1 (PD-L1) levels at both t0 and t3M which were significantly negatively associated with the mean SUVmax of the infarct at t3M. Conclusions: This is the first prospective study of serial 68Ga-DOTATATE PET/MRI in patients after MI. Our results indicate that 68Ga-DOTATATE PET is a useful tool for assessing post-infarct myocardial inflammation and that higher persistent 68Ga-DOTATATE PET signal at t3M may be linked to adverse remodelling outcomes. We further identify several biomarkers including serum PD-L1 levels at t0 and t3M as a potentially useful predictor of *in situ* myocardial inflammation as quantified by 68Ga-DOTATATE PET. ii) COVID-19 related myocardial injury and imaging Background: Cardiac injury is a well-recognised complication of acute COVID-19 infection, with varied aetiologies ranging from myocarditis to endothelial dysfunction, micro-embolic phenomena and acute coronary syndromes. While persistent cardiac symptoms in patients with post-acute COVID-19 syndrome (PACS) are also common, the underlying cause is less well understood. A variety of imaging abnormalities have been described on cardiac magnetic resonance imaging (MRI) in the post-COVID setting and immune system dysregulation has been implicated both in determining acute disease severity as well in the development of PASC. To our knowledge, only one previously published study has specifically examined the link between cardiac imaging abnormalities after COVID-19 infection and immune profiling of the affected individuals, which was also limited to quantification of peripheral immune cell populations. Aims: To examine cardiac involvement associated with COVID-19 infection using imaging and deep immunophenotyping employing mass cytometry by time-of-flight (CyTOF) as well as serum proteomic analysis. Methods: Participants who had a history of COVID-19 infection and suspected cardiac involvement but no prior history of myocardial infarction or heart failure and no history of recent systemic immunosuppression, were recruited between October 2020 and February 2022. Participants were recruited on the basis of either troponin I (TnI) elevation (>99th percentile upper reference limit), new onset heart failure not attributable to another cause or unexplained ongoing cardiac symptoms after their initial COVID-19 infection. Participants underwent full cardiac MRI with late gadolinium enhancement (LGE) imaging, peripheral blood cell immunophenotyping using mass cytometry by time-of-flight (CyTOF) and serum proteomic analysis (Olink® Target 96 Inflammation Panel). Results: 21 participants (mean age 47 (SD 13) years, 71% female) were enrolled, who either had PACS (n=17), suspected acute COVID myocarditis (n=2), or new-onset heart failure attributed to prior COVID infection (n=2). MRI showed a non-ischaemic pattern of LGE and/or visually overt myocardial oedema in 8 (38%) patients, including 5 (24%) with PACS. A further patient had impaired ventricular function in the absence of LGE. In total, 9 (43%) patients had MRI abnormalities defined as either a non-ischaemic pattern of LGE, oedema or ventricular impairment. Participants with MRI abnormalities exhibited differences in CyTOF and proteomic biomarker expression compared with participants without such abnormalities, including: increased CCL3, CCL4, CCL7 (MCP-3), CXCL1, FGF21, FGF23, IL-13, PD-L1 and ST1A1 levels, as well as CD4+ Th2-like cells; but decreased CD8+ T effector memory cells, including PD1+ CD8+ T cells and CD8+ αβ T cells. Using Lasso regression analysis including all baseline clinical characteristics, higher CCL7 (MCP-3) levels and lower CD8+ TEM cells were the strongest predictors of an abnormal MRI finding, with a composite AUC of 0.96. Conclusions: Cardiac involvement after COVID-19 infection in patients with MRI abnormalities is associated with CCL7 (MCP-3) elevation, a chemokine known to be important in viral myocarditis, as well as decreased CD8+ T effector memory cells. These findings potentially give insight into the pathogenesis of cardiac-specific involvement post-COVID-19.Item Open Access Investigating iPSC-Derived Macrophage Stimulation with Oxidised Low-Density Lipoprotein as an Effective In Vitro Model of Foam Cells in AtherosclerosisImaz, MariaOne of the earliest changes found in vessel walls at predilection sites for atherosclerotic plaque formation involves the retention and modification of lipoprotein particles. Macrophages and vascular smooth muscle cells (VSMCs) in the arterial wall respond by ingesting large quantities of the modified lipoproteins, such as oxidised low-density lipoproteins (oxLDLs), which are then metabolised to release cholesterol and cholesterol esters for use or recycling. Chronic uptake of modified particles eventually reduces the ability of macrophages and VSMCs to effectively break down and release the lipoprotein metabolites, causing intracellular accumulation of lipids. Existing studies have described potential foam cell-like populations using single cell RNA sequencing (scRNA-seq) of plaques, however due to known limitations of whole cell and more recent findings expanding our current knowledge of foam cell biology, much remains to be explored. Foam cells can be generated in vitro by lipid loading with modified lipoproteins or cholesterol, however, with a growing appreciation for the complexity of foam cells in vivo there remains uncertainty in which aspects of foam cell formation can be recapitulated with such models. To overcome some of the known drawbacks of single cell isolation, single nuclei extraction was optimised and applied to the selected vascular samples. One novel single nuclei and two existing single cell plaque RNA-seq datasets were analysed, showing successful annotation of expected plaque cell types and identification of individual foam cell populations, which demonstrated that single nuclei sequencing can be applied to vascular samples to describe their cellular heterogeneity. Intriguingly, a population of potential VSMC-derived foam cells could be seen to localise with macrophages when visualising cell populations in the single nuclei dataset, which had not been observed in existing single cell datasets. To identify foam cells in early stages of plaque development, I assessed the lipid content and macrophage and VSMC content of organ donor aortic samples across a range of tissue phenotypes to selected foam cell-rich samples that were used to generate single nuclei RNA-sequencing libraries. In order to compare gene profiles of in vivo foam cells to in vitro-generated foam cells, a bulk RNA-seq quality control and differential expression pipeline was established and applied to a large dataset that had been previously generated from a iPSC-derived macrophage foam cell model. OxLDL-treated macrophages expressed a number of expected foam cell gene markers, independent of M0, M1 and M2 polarisation states, however, *TREM2* was notably not differentially expressed in treated conditions. *TREM2* is commonly associated with foamy macrophages in vivo; this observation therefore highlighted that significant differences in expression profiles may preclude in vitro models from effectively modelling certain pathways in foam cell development. Comparison of in vivo foam cell gene profiles with in vitro macrophage foam cells showed unexpectedly little overlap. However, the analysis revealed that comparing bulk and single cell RNA-seq foam cell profiles was highly challenging, and required further evaluation to understand which aspects of foam cell formation can be explored in vitro. Future analysis of the data generated from arteries showing early signs of disease will serve to understand whether the in vitro conditions provide a more suitable model for initial foam cell formation.Item Embargo Characterisation of BMP Processing and Signalling in AdipocytesConstant, BenjaminAdipose tissue plays a crucial role in systemic energy homeostasis, and its dysfunction contributes to obesity and related cardiovascular diseases. Bone morphogenic protein (BMP) 8B, part of the Transforming Growth Factor-β (TGF-β) superfamily, is key in regulating energy balance via activating brown adipose tissue (BAT) thermogenesis, presenting a potential target for obesity treatment. However, the molecular mechanisms underlying BMP8B function in BAT remain unclear. It is reported that BMP8B signals through both SMAD1/5/8 and SMAD2/3 pathways in several cell types, but the impact of such unusual signalling specificity in adipocyte function is unknown. Thus, investigating BMP8B functions is crucial, both for therapeutic and molecular biology insights. My PhD project aims to elucidate the molecular mechanisms of BMP8B processing, regulation and signalling in adipocytes. My findings indicate that BMP8B secretion by mammalian cells is limited, partly due to interactions with cell-surface and matrix-bound extracellular matrix proteins like proteoglycans. This interaction likely involves the prodomain, as substituting BMP8B's prodomain with that of BMP10 enhances secretion. ProBMP8B is mainly cleaved at a consensus pro-protein convertase site near the mature ligand domain (R260TRR263↓), with alternative cleavage sites used when the primary site is unavailable, suggesting possible alternative processing of BMP8B. BMP8B from three commercial sources does not elicit either SMAD1/5 or SMAD2/3 phosphorylation in adipocytes, whereas BMP8B I produced in-house activates the SMAD1/5/8 but not the SMAD2/3 pathway. This activity was confirmed not to result from contamination with other TGF-β ligands. Preliminary data indicates that BMP8B's function in brown adipocytes is mediated through the type I receptor ALK3 and can be inhibited by the extracellular BMP ligand trap, Crossveinless 2. In my final year, my thesis expanded to compare BMP4, BMP9, and BMP10 signalling in adipocytes. BMP4 has a well-documented function in adipocytes, whereas roles for BMP9 and BMP10 are less known. Despite BMP9 and BMP10 having almost identical signalling activities in vascular endothelial cells, my results reveal distinct differences in the potencies and activities of these ligands in brown and white adipocytes. RNA sequencing (RNAseq) was performed to compare BMP4, BMP9, and BMP10 signalling in fully differentiated brown and white adipocytes, and the results show that these BMPs regulate shared BMP/SMAD and PPARγ target genes. However, their effects are distinct, influencing genes related to lipogenesis, adipogenesis, neurogenesis, and angiogenesis. Interestingly, I found that Endoglin, a known co-receptor for BMP9 and BMP10 in endothelial cells, is also highly expressed in adipocytes. Knocking out Endoglin in mouse primary brown and white preadipocytes reduced BMP9 and BMP4 signalling, uncovering a potential new aspect of BMP signalling regulation in adipocytes. In conclusion, the results presented in this thesis improve our understanding of the molecular mechanisms orchestrating BMP8B function in adipocytes, while also highlighting distinct functional roles of BMP4, BMP9, and BMP10, offering new therapeutic avenues for obesity and cardiometabolic disorders.Item Open Access Item Embargo Intestinal challenges shape the polarisation of dural meninges memory CD4+ T cellsFleming, AaronThe central nervous system (CNS) and the barriers that protect it, including the three-layered meninges, have been thought to be mostly devoid of immune cells and to interact sparingly with the peripheral immune system. However, recent work has shown that a diverse array of both innate and adaptive immune cells populate distinct niches of the brain and its borders and respond to insults from both within, and outside of, the CNS. The dura mater is the thickest and outermost skull-adjacent layer of the meninges, the membranes surrounding the brain and spinal cord, and has been shown to be particularly enriched in immune cell networks. Our lab has previously shown that the humoral immune landscape of the dura is intimately associated with that of the intestinal tract, in part reflecting the need of the dura to defend the underlying brain from intestinally derived blood-borne threats. Adaptive CD4+ T cells have been shown to populate the dura mater, but their phenotypic or clonal relationship to gut counterparts or role in local anti-pathogenic immune responses has been less studied. Using mouse models, we show that dural CD4+ T cell effector states are highly responsive to intestinal perturbations. We show that dural CD4+ T cell polarisation to a T-helper (Th)1, Th2, or Th17 state is predicated on the prevailing intestinal challenge, including bacterial and parasitic infections, as well as chemical-induced colitis. Using pathogens expressing specific model antigens, we show that the dural CD4+ T cell population contains cells bearing T-cell receptors (TCRs) specific for gut pathogens. This accumulation of dural antigen-specific CD4+ T cells depended, at least in part, on a CXCR6-CXCL16 axis. These dural antigen specific CD4+ T cells are long-lived and remain capable of a recall response following intravenous re- challenge for months after the initial oral infection. Using single-cell RNA sequencing, we identified clonal expansion of a population of dural CD4+ T cells expressing markers of tissue- residency. Finally, we show that dural CD4+ T cells induced by intestinal infection may help defend the brain against invasion by intravenous pathogens. Our work further delineates the intimate relationship between gut and dural immunity and reveals that CD4+ T cells form an integral component of the gut-dura axis. Further interrogation of the links between gut and dural CD4+ T cells may lead to a better understanding of how this axis may be therapeutically manipulated to promote host protection against infectious diseases of the CNS.Item Restricted New approaches and therapeutics for antimicrobial resistant Klebsiella: A comprehensive, multimodal approachZellmer, Caroline[Restricted]Item Embargo Cells of the Developing Human Heart and Great VesselsBayraktar, SemihThe human heart and the great vessels comprise various cell types that collectively support the heart’s homeostasis. Single-cell genomics has expanded our knowledge of the heart’s cellular heterogeneity. By gaining a deeper understanding of its cellular makeup, we can uncover how these cells converge to define cardiac identity and function and how perturbation to cellular identities can lead to diseases. This dissertation focuses on generating a high-resolution atlas of the first and second-trimester human heart and great vessels using single-cell genomics. The atlas contains 63 cell types with distinct identity, function, or location-specific signatures, such as the cardiomyocytes of different chambers and the vessel components specific to coronary or great vessels. Alongside molecular profiling, cellular communication mechanisms and gene regulatory networks are investigated, collectively aiming to advance *in vitro* models of cardiac cells. Chapter 1 introduces the topics covered throughout the thesis. After describing the materials and methods utilised in Chapter 2, an overview of the atlas is provided in Chapter 3. This is followed by a description of the individual cell types in subsequent chapters based on the macro anatomical structures they are associated with. Chapter 4 focuses on cardiomyocytes, which constitute the majority of the myocardium. In Chapter 5, the focus shifts to the vessels, encompassing the coronary and the great vessels. Chapter 6 explores the pericardium and endocardium as the two layers enveloping the myocardium. Ultimately, through the profiling obtained from the atlas, Chapter 7 proposes a framework to generate enhanced stem cell models to mimic the corresponding cells *in vivo*.Item Open Access The Epicardium as the Conductor of Cardiovascular Cells in Cardiac RegenerationRericha, PatrickStem cell therapy for cardiac repair is a field of regenerative cardiovascular medicine that is emerging as a promising therapeutic alternative to heart transplantation in patients suffering from chronic heart failure. Although promising, challenges to cell therapy for cardiac repair remain, including cell survival, maturation, graft size, revascularisation, and immunogenicity. It has recently been shown that co-transplantation of human embryonic stem cell (hESC)-derived epicardial cells (EPI) with hESC-derived cardiomyocytes (CM) improves cardiac repair processes with respect to engraftment, cell maturation, and graft and host vascularisation. One plausible explanation for the observed benefits of co-transplantation is paracrine-mediated effects of the epicardium towards CMs and endothelial cells (EC). While RNA sequencing data has been used to identify putative players in hESC-EPIs, little is known about their involvement in mediating hESC-CM maturation and angiogenesis. Here, the role of paracrine signalling in hESC-EPI-mediated promotion of hESC-CM maturation and angiogenesis is being investigated. To study the relative importance of secreted factors and extracellular vesicles (EVs) a protocol was established to split hESC-EPI conditioned medium into hESC-EPI-EVs and hESC-EPI-flowthrough, which is depleted in EVs. HESC-EPI-EVs failed to confirm the benefits on CM maturation observed in preliminary studies. Both fractions independently promoted tube formation in HUVECs *in vitro* and hESC-EPI-flowthrough additionally promoted proliferation and migration. Both fractions improved branching *in vivo* in chick yolk-sac membrane (YSM) assays. Characterisation of the hESC-EPI-EV-cargo on the protein and miRNA level lead to the identification of pro-angiogenic candidate factors that were screened *in vitro* and validated *in vivo*. Moreover, hESC-EPI-EVs reduced reactive oxygen species (ROS) in hESC-CMs and HUVECs *in vitro*. Viable mitochondria were transferred between hESC-EPIs and hESC-CMs and HUVECs, which was partly mediated by hESC-EPI-EVs. Identification of the paracrine signalling factors involved in the hESC-EPI-mediated promotion of CM maturation and revascularisation would make it possible to effectively address these challenges in stem cell therapy for cardiac repair, resulting in new therapeutic approaches.Item Embargo Domesticating the invader: how intronless genes escape HUSH silencingCohen-Gold, JonathanThe reverse flow of genetic information (the reverse transcription of RNA to DNA) allows retroviruses and retrotransposons to invade our genome. Together, retroelements make up more than 40% of the human genome. The HUSH epigenetic repressor complex defends the genome from retroelement invasion through the recognition and silencing of ‘intronless’ DNA, the hallmark of reverse transcription. Therefore, intronless cDNA provides the ‘abnormal molecular pattern’ which allows HUSH to distinguish foreign retroelements from host genes, making HUSH a part of the innate immune system. This presents a paradox: intronless genes (ILGs) comprise ~3-5% of the human protein-coding genome and are well expressed I.e. resistant to hush silencing despite themselves arising through retroelement invasion. I wanted to understand this paradox and determine how ILGs escape HUSH silencing. I initially assessed genome-wide RNA binding and transcriptional repression by HUSH to identify nucleic acid sequence determinants of HUSH-sensitivity. My findings suggest that ILGs evolve GC-rich sequence within their amino acid sequence constraints, through GC-biased codon usage and increased 5’UTR GC content. This enables their escape from HUSH despite the absence of introns, allowing beneficial retroelements to be domesticated. Decreasing GC content within an intronless coding sequence incrementally impaired recruitment of TREX, an essential mRNA export complex with a known GC binding RNA preference. This decreased GC content corresponded with reduced RNA export and increased HUSH repression. By tethering TREX components to intronless RNA, I could promote HUSH escape and this was most striking for ALYREF, an essential RNA- binding subunit of TREX which facilitates mRNA packaging. In contrast, depletion of TREX components (THOC1/2) induced HUSH-sensitivity of intronless reporters. As RNA binding by ALYREF and periphilin, the main RNA binding component of HUSH, is inversely proportional, I propose a model in which periphilin and ALYREF compete for nascent RNA binding. My results suggest this binding competition between periphilin and ALYREF determines the balance between HUSH-dependent silencing versus RNA nuclear export and gene expression.Item Embargo Mechanisms of Synthetic Lethality in Renal Cell CarcinomaBertlin, JamesClear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer, is characterised by biallelic inactivation of the von Hippel-Lindau (*VHL*) tumour suppressor gene, and inherited loss-of-heterozygosity of *VHL* underlies the eponymous hereditary cancer syndrome. The *VHL* protein product, pVHL, is the substrate recognition component of an E3 ubiquitin ligase complex responsible for targeting Hypoxia Inducible Factors (HIFs) for proteasomal degradation. Accumulation of the HIF-2α isoform, in particular, drives renal tumourigenesis. *VHL* loss occurs at an early stage of ccRCC development, often several years before the clinical manifestation of disease, and so is observed throughout the entire mass of both primary and metastatic tumours. Therapeutic approaches which specifically target cells harbouring *VHL* mutations could therefore provide effective anti-tumour responses with minimal off-target effects. In this thesis, I undertake CRISPR/Cas9 screens in ccRCC cell lines deficient in *VHL*, and in paired *VHL*-reconstituted cells, aiming to identify genetic vulnerabilities of cells lacking functional pVHL. These reveal a synthetic lethal relationship between Core Binding Factor β (CBF-β) and pVHL. While CBF-β is critical for normal osteogenesis and haematopoiesis, little is known about its role in renal tissue. CBF-β classically stabilises the Runt related transcription factors (RUNX1-3) to improve their transcriptional efficiency. However, using a variety of cell biology techniques, I demonstrate that knockout of *CBFB* is lethal in cells lacking *VHL* through a mechanism which is independent of both direct CBF-β-RUNX binding and the high HIF activity of *VHL*-null cells. In addition, RNA sequencing and mass spectrometry reveal that CBF-β depletion causes a widespread upregulation of interferon (IFN)-stimulated genes (ISGs), although this does not contribute to the cell death phenotype. Rather than occurring through a canonical IFN response, CBF-β loss-induced ISG expression relies on the direct transcriptional activity of IFN Regulatory Factor 3 (IRF3) and its stimulation via STING. Together, these results reveal a novel requirement of *VHL*-mutant ccRCC cells for CBF-β, loss of which provokes cell death and increases ISG expression. Besides triggering direct tumour cell killing, depletion of CBF-β may present a strategy to reactivate anti-tumour immune surveillance in renal cancer.Item Embargo Identification and validation of new therapeutic targets for squamous cell lung cancerKottmann, DanielLung squamous cell carcinoma (LUSC) is a subtype of non-small cell lung cancer and constitutes around 25% of all lung cancer cases worldwide. Despite recent advancements in cancer therapeutics, no targeted therapies have been approved specifically for LUSC. The lineage- survival oncogene and transcription factor SOX2 is dysregulated by gene amplification in a large proportion of LUSC tumours and has been implicated as a key driver in multiple preclinical functional studies. As a transcription factor, SOX2 itself makes a challenging drug target. New therapeutic approaches are required for this disease, and this project identified and evaluated potential therapeutic targets for SOX2-driven LUSC. Multi-omics data was used to identify drug target candidates from the SOX2 interactome in LUSC, and ten target candidates were selected for *in vitro* validation. Targeting the PI3K/AKT/mTOR pathway showed benefits in SOX2-driven disease, and the transcription factor FOXM1 was highlighted as a promising target candidate in SOX2-driven LUSC tumours. Additionally, transcriptional data of LUSC tumours was used to identify differentially expressed genes in SOX2-high tumours, and identified TrkB (encoded by the *NTRK2* gene) as a potential drug target candidate. Expression of TrkB correlated with SOX2 expression in squamous tumours, showed a functional role in squamous cancers, and *NTRK2* was confirmed as a transcriptional target of the SOX2 transcription factor. Deregulation of TrkB expression and specific transcript variants showed an impact on cell fitness, survival, and downstream signalling. While TrkB.T1 was shown to be the predominant transcript variant of the *NTRK2* gene in LUSC and other squamous cancers, both TrkB.T1 and TrkB.FL variants had a functional role in oncogenic signalling. Targeting TrkB in patients with high SOX2 expression might therefore be a promising strategy for further evaluation in LUSC. Finally, this project characterised current *in vitro* models of LUSC and undertook early efforts to develop new models from human bronchial epithelial cells. Better models are needed to study the disease and to test new therapeutics. Ultimately, good *in vitro* models of LUSC remain a challenge and require future work. In summary, this project identified a set of drug target candidates in SOX2-driven LUSC and presented *in vitro* validation results to support the further development of selected targets. This work generated new insights into SOX2 pathobiology in LUSC and contributed to the development of new therapeutic approaches to treat this disease with clear unmet medical needs.Item Embargo Contribution of Causal Aquaporin-1 Mutations to the Pathobiology of Pulmonary Arterial HypertensionWood, JenniferPulmonary arterial hypertension (PAH) is a rare and debilitating disease characterised by elevated pulmonary arterial pressure and extensive vascular remodelling in the lungs, with a lack of effective curative options. The identification of genetic variations in genes encoding components of the bone morphogenetic protein pathway in individuals with heritable PAH has opened up promising avenues for therapeutics, with encouraging results from preliminary clinical trials targeting this pathway. To deepen the understanding of the molecular mechanisms underpinning this rare condition, detect additional causative genes, and identify novel potential therapeutic strategies, a comprehensive study involving whole-genome sequencing was conducted on a cohort of over 1000 PAH patients. Within this cohort, rare causative heterozygous variants in the Aquaporin-1 (AQP1) gene were identified among a small subgroup of PAH patients. AQP1 is a water channel protein critical for maintaining endothelial cell integrity and fluid homeostasis. The primary aim of this thesis is to provide insight into the potential pathobiological significance of these rare AQP1 variants in PAH. Computational structural analysis using Pymol was employed to gain preliminary insights into the mutational effects of the identified variants. Subsequently, mutant AQP1 proteins were overexpressed in Hek293T cells, then subjected to confocal microscopy and membrane biotinylation followed by biotin-based ELISAs, to investigate whether the mutant proteins are able to traffic to the cellular membrane. These studies suggested normal membrane trafficking for the Arg126Cys, Val176Glu, Leu181Phe, and Arg195Trp AQP1 mutants, with comparable levels to that of wild-type AQP1. In contrast, the Trp213Term mutant is predicted to display dysfunctional trafficking, although further validation regarding this nonsense mutant is required. In addition, water and solute permeability through the wild-type and mutant AQP1 proteins was assessed using stable transfection of MDCK cells and transient transfection of Hek293T cells, respectively, followed by calcein self-quenching assays. None of the PAH-causative AQP1 mutants exhibited permeability to glycerol or urea, suggesting a retained ability for strict solute exclusion. Interestingly, water permeability was significantly reduced in the Arg126Cys AQP1 mutant, near to or completely abolished in the Val176Glu, Arg195Trp, and Trp213Term AQP1 mutants, but unaltered in the Leu181Phe AQP1 mutant. Finally, due to the recurrent nature of the Arg195Trp mutation, identified in 5 of 9 unrelated PAH patients with causal AQP1 variants, an Aqp1 R195W knock-in mouse model was generated to investigate its phenotypic effects *in vivo* and *ex vivo*. Surprisingly, neither heterozygous nor homozygous R195W mice displayed haemodynamic measurements suggestive of PH, either when exposed to normoxia or hypoxia. Nevertheless, the mutant mice exhibited significantly reduced urine osmolarity and signs of inflammation. Samples for future assessment of vascular remodelling through protein, mRNA, and immunohistochemistry analysis have been collected. In conclusion, this project has provided preliminary insights into the role of rare AQP1 variants in PAH and established crucial experimental protocols and reagents for further investigations. Ultimately, these findings may pave the way for the development of precision medicine approaches tailored to patients with specific AQP1 mutations, offering new hope for improved management and treatment outcomes in PAH.Item Embargo An unbiased proteomic approach to identifying cell surface markers in neutrophilsSubburayalu, Julien; Subburayalu, Julien [0000-0001-9243-0558]Introduction: Efferocytosis refers to the engulfment of dead cells by phagocytes such as neutrophils and macrophages and culminates in the beneficial removal of inflammatory cargo and auto-reactive material. While many of the mechanisms involved in efferocytosis have been described, the critically important ‘apoptotic-cell-associated molecular patterns’ have yet to be identified. Methods: Human whole blood samples were obtained from healthy volunteers. Neutrophils were isolated and subjected to a temperature shift to facilitate a wave of synchronised apoptosis. The plasma membrane proteome was assessed using tandem-mass-tag liquid chromatography-mass spectrometry (TMT-LC/MS) of apoptotic and time- and donor- matched non-apoptotic neutrophils, which allowed the identification of the plasma membrane proteins expressed or downregulated during apoptosis. In parallel, a method was developed to quantify efferocytosis. Briefly, apoptotic neutrophils or genome-modified neutrophil-like cells overexpressing the proteins identified to be up- regulated on apoptotic neutrophils, were fed to the monocytic cell line THP-1, which had been previously primed with phorbol-12-myristate-13-acetate (PMA) to acquire the functional properties of human macrophages. Efferocytosis was assessed by confocal microscopy and flow cytometry. Results: The late-stage apoptotic neutrophil plasma membrane profile identified several protein targets to be either up- or downregulated. The expression kinetics over time of ‘targets’ were then validated using flow cytometry. TIMD-4, a never previously described protein in (human) neutrophils, is actively up- regulated in preparation for apoptosis, and links to the phagocyte’s recognition and removal abilities of apoptotic cells, suggesting that TIMD-4 is a novel ‘eat me’ signal produced by apoptotic neutrophils. Conclusions: TMT-LC/MS can be successfully used to determine functionally relevant changes in the neutrophil plasma membrane, and has provided novel insights into the processes underlying efferocytosis of human neutrophils.Item Open Access Innate immunity in defence against infection and tissue repair of the urinary tractLoudon, KevinUrinary tract infections (UTI) are common and affect half of women and 5% of men during their lifetime. Most infections are caused by uropathogenic *Escherichia coli* (UPEC) and are limited to the lower urinary tract (cystitis), but ascending infection (pyelonephritis) can lead to fibrosis and the development of chronic kidney disease. A complex network of tissue-resident immune cells exists throughout the urinary tract that are vital for initiating and propagating local immune responses, however our understanding of their nature and function remains incomplete. In this thesis, we aim to understand two broad areas of tissue immunology: (i) the role of group 3 innate lymphoid cells (ILC3s) in bacterial cystitis and (ii) macrophage-derived amphiregulin (AREG) in acute pyelonephritis and infection-mediated scarring. Here, we identify type 17 immune response genes amongst the most up-regulated networks in the mouse bladder following UPEC challenge and identified ILC3s as a major source of these cytokines. Depletion of ILCs in Rag2-/- mice resulted in increased bladder bacterial load, identifying them as important players in bladder defence. Furthermore, we describe evidence of reciprocal cross talk between ILC3s and macrophages, with IL17 shaping bladder macrophage activation and polarisation. Together our findings reveal important insights into the orchestration and execution of type 17 immunity in bladder defence. AREG, is an epidermal growth factor receptor (EGFR) ligand that is expressed by a variety of activated immune cells, including macrophages and is a vital mediator of immune responses in infection and tissue repair. Using microarray analysis, we found AREG expression was higher in the renal medulla and augmented following UPEC stimulation. *In vivo, in vitro* and *in silico* experiments found renal macrophages to be a major source of AREG and mice globally deficient in *Areg* demonstrated increased susceptibility to pyelonephritis with impaired monocyte recruitment. Provisional findings in our haematopoietic- and myeloid-specific *Areg* knockout mice, also confirmed greater susceptibility to pyelonephritis. Furthermore, we describe accelerated repair of epithelial cells *in vitro* following the addition of recombinant AREG and conditioned medium from UPEC stimulated macrophages. Our data reveal a novel mechanism by which kidney MNPs interact with renal tubular cells to promote host-defence, organ integrity and repair in UTI. To study the role of AREG in infection-mediated kidney scarring we successfully developed a robust and reproducible *in vivo* murine model of chronic pyelonephritis. These experiments have found AREG expression to be highest in the medulla of kidneys with chronic pyelonephritis, and similarly enriched in medulla-derived macrophages. We found mice globally deficient in Areg were protected against kidney scarring and in 100% bone marrow chimeras, loss of AREG from the hematopoietic compartment offered greatest protection. Finally, using single-cell RNA sequencing we generate the first detailed atlas of the human kidney in chronic pyelonephritis and in conjunction with our murine data, offer insight into cellular mechanisms driving fibrosis, with the potential to inform strategies to reduce the development in CKD with patients with recurrent pyelonephritis.