Towards Metabolic Biomarkers for the Diagnosis and Prognosis of CKD

Chronic kidney disease, the gradual loss of renal function, is an increasingly recognized burden for patients and health care systems; globally, it has a high and rapidly growing prevalence, a significant mortality, and causes disproportionately high costs, particularly for hemodialysis and kidney transplantations. Yet, the available diagnostic tools are either impractical in clinical routine or have serious shortcomings preventing a well-informed disease management, although optimized treatment strategies with impressive benefits for patients have been established. Advances in bioanalytics have facilitated the identification of many genomic, proteomic, and metabolic biomarker candidates, some of which have been validated in independent cohorts. Summarizing the markers discovered so far, this chapter focuses on compounds or pathways, for which quantitative data, substantiating evidence from translational research, and a mechanistic understanding is available. Also, multiparametric marker panels have been suggested with promising diagnostic and prognostic performance in initial analyses, although the data basis from prospective trials is very limited. Large-scale studies, however, are underway and will validate certain sets of parameters and discard others. Finally, the path from clinical research to routine application is discussed, focusing on potential obstacles such as the use of mass spectrometry, and the feasibility of obtaining regulatory approval for metabolomics assays

Petri Net computational modelling of Langerhans cell Interferon Regulatory Factor Network predicts their role in T cell activation

Langerhans cells (LCs) are able to orchestrate adaptive immune responses in the skin by interpreting the microenvironmental context in which they encounter foreign substances, but the regulatory basis for this has not been established. Utilising systems immunology approaches combining in silico modelling of a reconstructed gene regulatory network (GRN) with in vitro validation of the predictions, we sought to determine the mechanisms of regulation of immune responses in human primary LCs. The key role of Interferon regulatory factors (IRFs) as controllers of the human Langerhans cell response to epidermal cytokines was revealed by whole transcriptome analysis. Applying Boolean logic we assembled a Petri net-based model of the IRF-GRN which provides molecular pathway predictions for the induction of different transcriptional programmes in LCs. In silico simulations performed after model parameterisation with transcription factor expression values predicted that human LC activation of antigen-specific CD8 T cells would be differentially regulated by epidermal cytokine induction of specific IRF-controlled pathways. This was confirmed by in vitro measurement of IFN-g production by activated T cells. As a proof of concept, this approach shows that stochastic modelling of a specific immune networks renders transcriptome data valuable for the prediction of functional outcomes of immune responses

Host-pathogen interactions between the human innate immune system and Candida albicans—understanding and modeling defense and evasion strategies

The diploid, polymorphic yeast Candida albicans is one of the most important humanpathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within thehuman host for a long time. Alterations in the host environment, however, can render C. albicansvirulent. In this review, we describe the immunological cross-talk between C. albicans and thehuman innate immune system. We give an overview in form of pairs of human defense strategiesincluding immunological mechanisms as well as general stressors such as nutrient limitation,pH, fever etc. and the corresponding fungal response and evasion mechanisms. FurthermoreComputational Systems Biology approaches to model and investigate these complex interactionare highlighted with a special focus on game-theoretical methods and agent-based models. Anoutlook on interesting questions to be tackled by Systems Biology regarding entangled defenseand evasion mechanisms is given

Critical roles of dendritic cells and macrophages in cardiovascular disease

Cardiovascular disease is a major cause of mortality in the world, and is rapidly increasing. To understand the inflammatory response of various diseases, we have made tremendous advances in molecular and cellular research to show clear evidence that macrophages and dendritic cells play central roles in cardiovascular diseases, such as atherosclerosis and myocardial infarction. We have identified the two conventional DCs subsets (cDC1 and cDC2) in heart of healthy mouse. As expected, we found that the IRF8-expressing cDC1, but not the IRF4-expressing cDC2, was dependent on Flt3 for its development. Myocardial infarction significantly increased the infiltration of CD45+ leukocytes in the infarcted heart, including macrophages, neutrophils and DCs. Among cDC, the most significantly increased subset was the cDC2. The diphtheria toxin receptor (DTR) mediated depletion of Zbtb46-expressing DCs in myocardial infarcted mice led to a significant decrease of IL-1β expression together with an increased recruitment of leukocytes. This dramatically reduced the infarcted size and improved cardiac function, suggesting that cardiac DCs play an important role in immune cell infiltration following myocardial infarction. In another study, we developed, for the first time, a lipid probe-based flow cytometry method to analyze foam cells in atherosclerotic aorta. Our method enables to isolate foamy and non-foamy macrophages in order to perform the transcriptomic analysis of these cells. In addition, this technique allows to assess the severity of atherosclerosis and the characteristics of foam cells. RNA-seq analysis showed that lipid-laden foamy macrophages in atherosclerotic lesions clearly expressed different transcripts compared to non-foamy macrophages. Surprisingly, we found that non-foamy macrophages showed a pro-inflammatory signature reflected by the up-regulation of cytokines, such as Il1β, Tnf and Nlrp3. However, foamy macrophages up-regulated genes related to the transport and uptake of lipids (cholesterol and fatty acid). Collectively, we specifically identified the function of dendritic cells and macrophages in the pathogenesis of cardiovascular diseases. Understanding the precise role of these cells will help to develop appropriate immune-therapeutic strategy to attenuate cardiovascular disease.Les maladies cardiovasculaires représentent un risque majeur de mortalité dans le monde et la prévalence de ces pathologies ne cesse d’augmenter. Pour comprendre la réponse inflammatoire de ces maladies, nous avons fait d’énormes progrès dans la recherche moléculaire et cellulaire afin de démontrer clairement que les macrophages et les cellules dendritiques jouent un rôle central dans l’athérosclérose et l’infarctus du myocarde, deux maladies cardiovasculaires mortelles. Nous avons trouvé que, à l’état normal, le coeur de souris abrite les deux types de cellules dendritiques classiques (cDC1 et cDC2). Conformément à ce qui est connue sur ces les cDC, nous avons constaté que les cDC1, qui expriment IRF8, dépendent du Flt3, un facteur de croissance important pour leur développement alors que les cDC2, qui expriment IRF4, sont Flt3 indépendantes. Nous avons trouvé que le nombre total leucocytes CD45+ augment dans le coeur des souris ayant subi l'infarctus du myocarde. Ces leucocytes étaient majoritairement des macrophages, des neutrophiles et des cDCs. Parmi ces dernières, les cDC2 ont connu une plus forte augmentation. Dans un premier projet, nous avons vérifié le rôle de ces cDCs dans cette pathologie. Pour ce faire, nous avons procédé à la déplétion de ces cellules en utilisant un modèle de souris exprimant le récepteur de la toxine diphtérique (DTR) au même temps que Zbtb46. L’expression spécifique de ce dernier dans les cDCs induit l’expression de la DRT et l’injection ultérieure de la toxine diphtérique provoque la déplétion des cDCs chez ces souris transgéniques. Nos travaux ont montré que la déplétion des cDCs diminue le recrutement des leucocytes et l'expression de la cytokine inflammatoire l'IL-1β dans le coeur des souris ayant subi l’infarctus du myocarde. Cela réduit considérablement l’ampleur de l'infarctus et améliore la fonction cardiaque, suggérant que les cDCs jouent un rôle important dans l'infiltration de cellules immunitaires après infarctus du myocarde. Dans une autre étude, nous avons mis au point, pour la première fois, une méthode basée sur l’utilisation de sonde lipidique pour analyser les cellules spumeuses de l'aorte par cytométrie en flux. Cette méthode permet de cibler et isoler les macrophages spumeux et non-spumeux afin d’analyser leur profile transcriptomique. En plus de caractériser ces cellules, cette technique permet d’évaluer la taille des plaques d’athérosclérose. Le séquençage des ARN (ARN-seq) a montré que les macrophages spumeux chargés de lipides dans les lésions athérosclérotiques expriment clairement différents transcrits par rapport aux macrophages non-spumeux. De manière surprenante, nous avons constaté que les macrophages nonspumeux présentaient une signature pro-inflammatoire reflétée par une régulation à la hausse de l’Il1β, le Tnf et Nlrp3. Par contre, les macrophages spumeux présentait un profile non inflammatoire caractérisé une augmentation de l’expression des gènes liés au transport et à l'absorption de lipides (cholestérol et acides gras). Contrairement à ce qui est connue jusqu’à maintenant sur le rôle pathogénique des macrophages spumeux, nos résultats montrent que ces cellules jouent un rôle plutôt protecteur dans l’athérosclérose. Collectivement, nous avons spécifiquement identifié la fonction des cDC et des macrophages dans la pathogenèse des maladies cardiovasculaires. Comprendre le rôle précis de ces cellules aidera à développer une stratégie immunothérapeutique appropriée pour atténuer les maladies cardiovasculaires

Metabolic Disturbance and Th17/Treg Imbalance Are Associated With Progression of Gingivitis

ObjectiveThis study sought to explore the role of metabolic disturbance in immunoregulation of gingivitis targeting T helper 17 cells (Th17)/regulatory T cell (Treg).Materials and MethodsA total of 20 gingivitis patients and 19 healthy volunteers were recruited. Quantitative real time polymerase chain reaction (qRT-PCR) was used to evaluate expression patterns of Forkhead box protein P3 (Foxp3), transforming growth factor-β (TGF-β), retinoid-related orphan receptor-gammat (RORγt) and interleukin 17A (IL-17A) in the peripheral blood lymphocytes of subjects across the two groups. Moreover, the enzyme-linked immunosorbent assay (ELISA) technique was used to detect levels of TGF-β, IL-4, IL-6,TL-10 and L-17A secreted in the plasma as well as the SIgA secreted in saliva. Flow cytometry was used to detect the percentage of CD4+CD25+ Foxp3+Treg cells and the percentage of CD4+IL-17A+ Th17 cells in whole blood of subjects in both groups. Gas chromatography-mass spectrometry (GC-MS) was employed to analyze the plasma metabolites in the gingivitis patient group. Statistical analysis was applied to determine whether the plasma metabolites and related metabolic pathways significantly differed between gingivitis patients and healthy controls. Ingenuity pathway analysis (IPA) was employed to identify the potential relation between the metabolites and the Th17 and Treg related pathway.ResultsThe percentages of CD4+IL17A+Th17 cells and IL-17 significantly increased in the peripheral blood in the gingivitis group. Moreover, the upregulation of IL-17A mRNA and RORγt mRNA were also found in the gingivitis group. However, the percentage of CD4+CD25+ Foxp3+Treg cells and Foxp3 mRNA in the whole blood did not significantly change. However, TGF-β mRNA as well as TGF-β, IL-4, IL-6, IL-10 in the periperial blood and SIgA in the saliva were higher in the gingivitis group. Notably, that the ratio of Th17/Treg cells was significantly increased during peripheral circulation. Furthermore, we identified 18 different metabolites which were differentially expressed in plasma between the gingivitis and healthy control groups. Notably, the levels of cholesterol, glycerol 1-octadecanoate, d-glucose, uric acid, cyclohexaneacetic acid, 3-pyridine, tryptophan, and undecane 2,4-dimethyl were significantly up-regulated. whereas the levels of lactic acid, glycine, linoleic acid, monopalmitic acid, glycerol, palmitic acid, pyruvate, 1-(3-methylbutyl)-2,3,4,6-tetramethylbenzene, 1 5-anhydro d-altrol, and boric acid were down-regulated in the gingivitis group, relative to healthy controls. IPA showed that these metabolites are connected to IL17 signaling, TGF-B signaling, and IL10 signaling, which are related closely to Th17 and Treg pathway.ConclusionOverall, these results showed that disturbance to glycolysis as well as amino and fatty acid metabolism are associated with Th17/Treg balance in gingivitis. Impaired immunometabolism may influence some periodontally involved systemic diseases, hence it is a promising strategy in targeted development of treatment therapies

Investigating a sustainable means of detecting and monitoring Alzheimer’s disease risk via non-invasive biomarkers

Compelling evidence has identified the kynurenine pathway (KP) as an avenue to predict, diagnose, and measure the progression of neurodegenerative diseases. This provides promising directions for the development of novel and precise treatment options for individuals experiencing metabolic dysregulation and cognitive challenges. However, the complex interrelationship between the KP and Alzheimer’s disease (AD) risk has not been characterised, in part due to a lack of understanding of: i) How normal KP function is characterised across the lifespan; ii) A lack of evidence from ‘at-risk’ cohorts; and iii) The heterogeneous and unvalidated methods used and the incomplete datasets available. This thesis will address these research gaps by developing and applying validated targeted metabolomics approaches to assess the diagnostic accuracy of the KP as a non-invasive biomarker for AD risk across various sample types and cohorts. Outcomes from this research aims to show or predict the relationship between the KP and AD risk. There are three aims to this thesis that will be addressed across three studies: Aim 1: Appraise and characterise the existing literature on how KP metabolites are measured and establish a set of normative data; Aim 2: Identify and develop a validated analytical method to quantify KP metabolites in the liquid biopsies of healthy adults in a range of human biofluids going beyond blood samples; and Aim 3: Analyse KP metabolites in the serum and urine of older adults at risk and not at risk of AD and assess their responsivity to treatment from clinical trial data. The overarching aim of this thesis is to profile a variety of non-invasive and minimally invasive biofluids from healthy adults across the lifespan, as well as older adults at risk of AD, to characterise the relationship between the KP and AD risk. The purpose of this is to seed new directions for pragmatic biomarker discovery in the fields of AD risk prediction and the monitoring of responsivity to treatment. As an additional note, this thesis will benefit from using analytical methods which have been objectively validated for diagnostic accuracy, and controlling for sex differences, lifestyle factors, and inter-sample variation

Cancer Immunology

The past decade has seen immunotherapy rise to the forefront of cancer treatment. This Special Issue of Cancers aims to elaborate on the latest developments, cutting-edge technologies, and prospects in cancer immunology and immunotherapy. Seventeen exceptional studies, including original contributions and review articles, written by international scientists and physicians, primarily concerning the fields of tumor biology, cancer immunology, therapeutics, and drug development, comprise the main body of this Special Issue