The role of the cytokines IL-17A and IL-33 in inflammatory arthritis and psoriasis

The inflammatory autoimmune diseases rheumatoid arthritis, psoriatic arthritis and psoriasis have seen a break through in therapy by targeting cytokines in the last decade. Interleukin-17A, a potential new target, is considered as a crucial player in rheumatoid arthritis, and has been suggested to be produced by CD4+ T cells (Th17 cells). I explored the cellular sources of IL-17A in human established RA synovium. Surprisingly, only a small proportion of IL-17 positive cells were T cells without expression of a Th17 marker CCR6. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. These data do not contradict a crucial role for IL-17A in RA pathogenesis, however, suggest that in addition to Th17 cells, cells of the innate immune system, particularly mast cells, may be an important component of the effector IL-17A response. Psoriasis is a common chronic autoimmune disease of the skin characterized by hyperplasia of epidermal keratinocytes with associated inflammation. IL-33 is a new member of the IL-1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently broader immune potential has been discovered for IL-33 particularly via mast cell activation. With its expression at body barrier surfaces it is assumed to act as an alarmin. In this thesis I demonstrate that IL-33 expression is up-regulated in the epidermis of psoriatic lesions, compared to healthy skin, thus indicating that IL-33 may be a mediator regulating crosstalk between keratinocytes and infiltrating immune cells in psoriatic plaques. In a phorbol ester-induced model of skin inflammation ST2-/- mice exhibited reduced cutaneous inflammatory responses compared to WT mice. Furthermore, consecutive injections of IL-33 into the ears of mice induced a psoriasis-like inflammatory lesion. This was partially mast cell dependent and cellular analysis demonstrated recruitment of neutrophils to the ear. This concludes that IL-33, via activation of mast cells and recruitment of neutrophils, may play a role in psoriasis plaque inflammation. In the last part of the thesis I tested if nanoparticles can be utilized to image cytokine driven inflammation. Bio-linkages with protein-nanoparticles have been established and in vivo detection of nanoparticles performed. This final interdisciplinary outlook demonstrates a still to be established/finalized method with great potential

Adipose derived stromal vascular fraction: therapeutic potential of renal artery administration in renal ischaemia reperfusion injury

Shortages of kidneys for transplantation is a universal concern. Countries like the United Kingdom have had success improving the number of kidneys available notably through expanding their living donation program. However, trends indicate the discrepancy between supply and demand will never fully be met with current practise. Bioengineering of donor specific organs may provide the ultimate answer, but despite rapidly growing progress it will be many more years until complex organs, such as a kidney, are available for transplantation. Utilising kidneys that traditionally would not be considered suitable for transplantation will drastically increase the donor pool of kidneys. To implant these ‘marginal’ kidneys we have to minimise the ischaemia reperfusion injury (IRI) they sustain during the transplantation process. Cellular therapies offer the greatest potential in ameliorating transplant related IRI and allowing these marginal kidneys to recover with acceptable long-term function. The field of cellular therapies, in particular stem cell therapy, has expanded rapidly in the last ten years. Adipose tissue offers one of the most attractive sources of stem cells due to its relatively easy accessibility and high abundance of stem cells. The adipose derived stromal vascular fraction (ADSVF), which contains the adipose derived stem cells, can quickly be extracted from the adipose tissue with minimal technical expertise. Multiple animal studies of renal IRI have demonstrated significantly improved kidney function after treatment with ADSVF. However, administration of freshly isolated, uncultured ADSVF administered via the renal at the time of transplantation has never been attempted in human kidneys. Before considering clinical translation, we need to better understand the use of ADSVF in such settings. In chapter one, experiments characterise the ADSVF obtained from inguinal and peri-renal adipose tissue of both rats and humans. Chapter two describes the development of a novel animal model which closely mimics the transplant recipient in order to accurately investigate the ADSVF. Chapter three demonstrates the biodistribution of the ADSVF administered via the renal artery and chapter four highlights some of the potential mechanisms of action of the ADSVF. In summary, ADSVF from peri-renal and inguinal adipose tissue consist of a similar heterogenous cell population, although they differ in the proportions of each subset. When administered via the renal artery, ADSVF likely ameliorate IRI via multiple mechanisms, by reducing the immediate injury, modulating the inflammatory response and reducing the progression to fibrosis. Administration via the renal artery is an effective means of reducing non-target biodistribution and deliver the majority of the ADSVF to the cortex of the kidney. The animal model developed as part of this research better mimics the transplant patient, compared to existing models, without increasing the risks to the animal. The findings of this research combined with recently published data using mesenchymal stem cells in human kidney IRI, provides enough support to consider renal artery administration of uncultured ADSVF. Initially this translational research could be performed on declined kidney grafts

The expression and function of the mucin-like glycoprotein podoplanin in glioblastoma

The mucin-like sialoglycoprotein podoplanin (PDPN) is widely expressed throughout the human and rodent body. Although numerous studies have revealed its essential function in development, especially of the lymphatic system, the lungs and heart, the overall picture of its physiologic function is still incomplete. Emerging evidence of the past decade has associated PDPN de novo or overexpression with numerous cancer entities including glioblastoma, and in particular with the invasive behavior of tumor cells. As the infiltrative growth of tumor cells is one major challenge in glioblastoma therapy, the identification of novel candidates in tumor cell migration remains an essential pre-requisite for the development of new and effective therapeutic means. However, the postulated pro-tumorigenic and pro-invasive function of PDPN in glioblastoma has never been validated in vivo. Moreover, the underlying mechanism of a potential malignant effect of PDPN has not been addressed. Thus, the aim of this study was to close this gap of knowledge by the combination of correlative and functional assays. Descriptive in vivo approaches involving patient-derived xenografts were primarily taken to confirm the previous correlations of PDPN expression and malignant progression and to establish a model that enables the investigation of underlying mechanisms. For the functional validation of the hypothesis that PDPN is a major driver of glioblastoma progression and especially invasion, the gene was deleted by the novel CRISPR/Cas9 technology. Xenotransplantations of control and knockout cells indicated the dispensability of PDPN for glioblastoma growth and progression. The reliable analysis of the postulated pro-invasive function of PDPN required the optimization of a three-dimensional invasion assay based on organotypic brain slice cultures. The usage of adult murine brain slices and a red emitting fluorescent membrane dye significantly improved the assay quality. The application of this advanced technique identified PDPN as a non-rate limiting component in glioblastoma cell invasion. These data and the detailed analysis of further malignant features including proliferation, apoptosis and angiogenesis have rebutted the previous assumption of a tumor promoting effect of PDPN. Despite the dispensability of PDPN for tumor development and tumor cell invasion, the obtained results suggest PDPN as a marker for malignant glioblastoma cells. In conclusion, this study represents an important contribution in the process of preclinical drug development, as the results object the frequently suggested development of a PDPN blocking therapeutic agent. Instead, this work suggests PDPN as a marker for prognosis or targeted delivery of cytotoxic compounds into glioblastoma tumor cells

Microencapsulated human islets as a therapy for type 1 diabetes

Microencapsulating human islets is a means being explored to overcome the immune mediated destruction of the graft without toxic immunosuppression. Despite promising studies in various animal models, encapsulated human islet transplantation has so far not made an impact in the clinical setting. Many non-immunological and immunological factors such as limited islet availability, reduced immunoprotection, effect of the encapsulation process, hypoxia and inflammatory response hinder the successful application of this promising technology. In this study, strategies were designed to overcome the above factors which should enhance the survival and function of encapsulated human islets. The study demonstrated that human islets can be shipped safely over long distances and encapsulated without affecting cell survival and function, thus overcoming its limited availability. A strategy to reduce the pore size of the microcapsules by increasing the gelling time was unsuccessful and produced brittle capsules. The microcapsules produced were stable and biocompatible in rodents but not in the baboon. The encapsulation process had little or no effect on the cellular transcriptome of human islets and on their ability to function both in vitro and in vivo. The study showed that encapsulated human islets placed in the peritoneal cavity experience hypoxia in the immediate post-transplantation period. Pre-treating encapsulated islets with desferrioxamine enhanced expression of hypoxia inducible factor-1α and vascular endothelial growth factor, and reduced the number of encapsulated islets needed to normalize blood glucose levels from 2,000 to as few as 750 IEQs. Allo- and xeno- transplantation of encapsulated insulin producing cells into immunocompetent rats resulted in a dense pericapsular fibrotic overgrowth. Coating heparin on the microcapsules reduced the fibrotic overgrowth in the allo- but not the xeno- graft model. The absence of dense pericapsular fibrotic overgrowth in the allografted humanized mouse suggests that accelerated efforts are needed to develop a suitable preclinical animal model. In summary, this thesis shows that microencapsulating human islets is safe and illustrates strategies to overcome hypoxia and reduce pericapsular fibrosis. However, further modifications to the microcapsule are required to prevent the fibrotic overgrowth, if encapsulated human islets or beta cell surrogates are to become a viable option as a therapy for type 1 diabetes in humans

Molecular and cellular pathogenesis of the erythroid defect and the therapeutic effect of glucocorticoids in Diamond-Blackfan anaemia

Diamond-Blackfan Anaemia (DBA) is a rare inherited bone marrow (BM) failure syndrome characterised by selective anaemia, congenital anomalies and predisposition to malignancy. DBA is caused by monoallelic, loss-of-function mutations in ribosomal protein (RP) genes. Delineation of the precise erythroid defect underpinning anaemia in DBA has been hampered by a lack of markers that define cells giving rise to burst- and colony-forming unit-erythroid (BFU-E and CFU-E) colonies, i.e., the clonogenic assays that quantify early and late erythroid progenitor (EP) potential respectively. By combining flow-cytometry, cell-sorting and single cell clonogenic assays, I identify Lin-CD34+CD38+CD45RA-CD123-CD71+CD41a-CD105-CD36- BM cells as early EP (EEP) and Lin-CD34+/-CD38+CD45RA-CD123-CD71+CD41a-CD105+CD36+ cells as late EP (LEP) giving rise to BFU-E and CFU-E respectively. By applying these definitions to DBA, I reveal, for the first time, that both quantitative and qualitative defects in EEP and LEP contribute to defective erythropoiesis in DBA and its restoration by corticosteroids (CS). I also demonstrate that, while an EP defect associated with profound loss of erythroblasts (EB) is present in transfusion-dependent (TD) patients with RPS gene-associated DBA, EP/EB are relatively preserved in TD patients with RPL gene variants. Transcriptome profiling by RNA-sequencing of FACS-purified EB from RPL genotype patients compared with age-matched controls, provides a unique dataset showing a distinct DBA molecular signature, characterised by p53 and inflammatory signalling pathways. Remarkably, the cellular differences between RPL and RPS gene-associated DBA correlate with divergent clinical phenotypes: patients with RPL rather than RPS gene pathogenic variants are more likely to present with anaemia at an older age, and to respond to CS. Finally, although DBA is rare, I demonstrate its wider utility as a paradigm of disordered erythroid commitment, providing insight into normal haematopoiesis. The strategies described for prospective isolation of EP/EB constitute an original contribution to knowledge that will facilitate more incisive study of normal and aberrant erythropoiesis.Open Acces

The role of the cytokines IL-17A and IL-33 in inflammatory arthritis and psoriasis

The inflammatory autoimmune diseases rheumatoid arthritis, psoriatic arthritis and psoriasis have seen a break through in therapy by targeting cytokines in the last decade. Interleukin-17A, a potential new target, is considered as a crucial player in rheumatoid arthritis, and has been suggested to be produced by CD4+ T cells (Th17 cells). I explored the cellular sources of IL-17A in human established RA synovium. Surprisingly, only a small proportion of IL-17 positive cells were T cells without expression of a Th17 marker CCR6. Unexpectedly, the majority of IL-17A expression colocalized within mast cells. These data do not contradict a crucial role for IL-17A in RA pathogenesis, however, suggest that in addition to Th17 cells, cells of the innate immune system, particularly mast cells, may be an important component of the effector IL-17A response. Psoriasis is a common chronic autoimmune disease of the skin characterized by hyperplasia of epidermal keratinocytes with associated inflammation. IL-33 is a new member of the IL-1 superfamily that signals through the ST2 receptor and was originally defined as an inducer of T helper 2 (Th2) cytokines. Recently broader immune potential has been discovered for IL-33 particularly via mast cell activation. With its expression at body barrier surfaces it is assumed to act as an alarmin. In this thesis I demonstrate that IL-33 expression is up-regulated in the epidermis of psoriatic lesions, compared to healthy skin, thus indicating that IL-33 may be a mediator regulating crosstalk between keratinocytes and infiltrating immune cells in psoriatic plaques. In a phorbol ester-induced model of skin inflammation ST2-/- mice exhibited reduced cutaneous inflammatory responses compared to WT mice. Furthermore, consecutive injections of IL-33 into the ears of mice induced a psoriasis-like inflammatory lesion. This was partially mast cell dependent and cellular analysis demonstrated recruitment of neutrophils to the ear. This concludes that IL-33, via activation of mast cells and recruitment of neutrophils, may play a role in psoriasis plaque inflammation. In the last part of the thesis I tested if nanoparticles can be utilized to image cytokine driven inflammation. Bio-linkages with protein-nanoparticles have been established and in vivo detection of nanoparticles performed. This final interdisciplinary outlook demonstrates a still to be established/finalized method with great potential.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Single stem cell clones of an acute myeloid leukaemia patient display functional heterogeneity in vivo

Acute myeloid leukaemia (AML) is a haematopoietic malignancy characterised by major genetic and functional heterogeneity. As clinical challenge, the most adverse subclone determines a patient’s outcome. A better understanding of tumour heterogeneity is required to direct treatment strategies against adverse subclones. In the present work, I aimed at characterising the genetic, epigenetic, transcriptomic and functional heterogeneity within the tumour sample of a single patient with AML. To enable functional in vivo studies, a patient-derived xenograft (PDX) mouse model was established from the AML sample. Upon re-transplanting minor numbers of PDX AML cells into immunocompromised mice, I generated twelve PDX AML populations that derived from a single stem cell, as proven by molecular barcoding using lentiviruses. The resulting PDX AML single cell clones (SCCs) were lentivirally marked with an individual combination of fluorochromes, enabling their separate analysis via flow cytometry in competitive in vivo assays. Epigenetic and transcriptomic analyses showed that PDX AML SCCs clustered according to their origin from first or second relapse. Genetic analyses revealed the existence of at least four genetically distinct subclones. Functional in vivo assays uncovered heterogeneity between the different PDX AML SCCs concerning stem cell capacity, growth, dormancy and treatment response. The most adverse PDX AML SCC displayed rapid growth in competitive in vivo assays combined with a partial resistance towards treatment with conventional chemotherapy. The aggressive functional behaviour was associated with a unique deletion of chromosome 17q correlating to i.a. an enrichment in HOX signalling. Of clinical importance and while resistant towards several treatment options, the clone responded to systemic treatment with the hypomethylating agent 5-azacitidine. Taken together, the data revealed that the known heterogeneity within tumor cells of a single patient with AML results in major functional heterogeneity in vivo. Clonal evolution of genetic changes can generate functionally aggressive clones, which might still respond to well-chosen second-line treatment, improving the clinical situation

Innovative Strategies in Tissue Engineering

In spite of intensive investments and investigations carried out in the last decade, many aspects of the stem cell physiology, technology and regulation remain to be fully defined. After the enthusiasm that characterized the first decade of the discovery that when given the right cue, stem cells could repair all the different tissues in the body; it is now time to start a serious and coordinated action to define how to govern the stem cell potential and to exploit it for clinical applications. This can be achieved only with shared research programs involving investigators from all over the world and making the results available to all.The Disputationes Workshop series (http://disputationes.info) is an international initiative aimed at disseminating stem cell related cutting edge knowledge among scientists, healthcare workers, students and policy makers. The present book gathers together some of the ideas discussed during the third and fourth Disputationes Workshops held in Florence (Italy) and Aalborg (Denmark), respectively. The aim of this book is to preserve those ideas in order to contribute to the general discussion on organ repair and to bolster a fundamental scientific and technological leap forwards the treatment of otherwise incurable diseases

Innovative Strategies in Tissue Engineering

In spite of intensive investments and investigations carried out in the last decade, many aspects of the stem cell physiology, technology and regulation remain to be fully defined. After the enthusiasm that characterized the first decade of the discovery that when given the right cue, stem cells could repair all the different tissues in the body; it is now time to start a serious and coordinated action to define how to govern the stem cell potential and to exploit it for clinical applications. This can be achieved only with shared research programs involving investigators from all over the world and making the results available to all.The Disputationes Workshop series (http://disputationes.info) is an international initiative aimed at disseminating stem cell related cutting edge knowledge among scientists, healthcare workers, students and policy makers. The present book gathers together some of the ideas discussed during the third and fourth Disputationes Workshops held in Florence (Italy) and Aalborg (Denmark), respectively. The aim of this book is to preserve those ideas in order to contribute to the general discussion on organ repair and to bolster a fundamental scientific and technological leap forwards the treatment of otherwise incurable diseases