Early Detection of Peripheral Blood Cell Signature in Children Developing Beta-Cell Autoimmunity at a Young Age

The appearance of Type 1 diabetes (T1D)-associated autoantibodies is the first and only measurable parameter to predict progression toward T1D in genetically susceptible individuals. However, autoantibodies indicate an active autoimmune reaction, wherein the immune tolerance is already broken. Therefore, there is a clear and urgent need for new biomarkers that predict the onset of the autoimmune reaction preceding autoantibody positivity or reflect progressive beta-cell destruction. Here we report the mRNA-sequencing-based analysis of 306 samples including fractionated samples of CD4+ and CD8+ T cells as well as CD4-CD8- cells fractions and unfractionated PBMC samples longitudinally collected from seven children that developed beta-cell autoimmunity (Cases) at a young age and their matched controls. We identified transcripts, including interleukin-32 (IL32) that were upregulated before T1D-associated autoantibodies appeared. Single-cell RNA-seq studies revealed that high IL32 in Case samples were contributed mainly by activated T cells and NK cells. Further, we showed that IL32 expression can be induced by a virus and cytokines in pancreatic islets and beta-cells, respectively. The results provide a basis for early detection of aberrations in the immune system function before T1D and suggest a potential role for IL32 in the pathogenesis of T1D.</p

Studies of Enterovirus Infection and Induction of Innate Immunity in Human Pancreatic Cells

Several epidemiological and clinical studies have indicated a possible role of Enterovirus (EV) infection in type 1 diabetes (T1D) development. However, the exact casual mechanism of these viruses in T1D development is not known. The aim of this thesis is to study various EVs that have been shown to differ in their immune phenotype, lytic ability, association with induction of islet autoantibodies, ability to replicate, cause islet disintegration and induce innate antiviral pathways in infected pancreatic cells in vitro. Furthermore, EV presence and pathogenic process in pancreatic tissue and isolated islets of T1D patients was also studied. Studies in this thesis for first time show the detection of EV RNA and protein in recent onset live T1D patients supporting the EV hypothesis in T1D development. Further all EV serotypes studied were able to replicate in islets, causing variable amount of islet disintegration ranging from extensive islet disintegration to not affecting islet morphology at all. However, one of the EV serotype replicated in only two out of seven donors infected, highlighting the importance of individual variation between donors. Further, this serotype impaired the insulin response to glucose stimulation without causing any visible islet disintegration, suggesting that this serotype might impaired the insulin response by inducing a functional block. Infection of human islets with the EV serotypes that are differentially associated with the development of islet autoantibodies showed the islet cell disintegration that is comparable with their degree of islet autoantibody seroconversion. Suggesting that the extent of the epidemic-associated islet autoantibody induction may depend on the ability of the viral serotypes to damage islet cells. Furthermore, one of the EV strains showed unique ability to infect and replicate both in endo and exocrine cells of the pancreas. EV replication in both endo and exocrine cells affected the genes involved in innate and antiviral pathways and induction of certain genes with important antiviral activity significantly varied between different donors. Suggesting that the same EV infection could result in different outcome in different individuals. Finally, we compared the results obtained by lytic and non lytic EV strains in vitro with the findings reported in fulminant and slowly progressing autoimmune T1D and found some similarities. In conclusion the results presented in this thesis further support the role of EV in T1D development and provide more insights regarding viral and host variation.  This will improve our understanding of the possible causative mechanism by EV in T1D development

Studies of Enterovirus Infection and Induction of Innate Immunity in Human Pancreatic Cells

Several epidemiological and clinical studies have indicated a possible role of Enterovirus (EV) infection in type 1 diabetes (T1D) development. However, the exact casual mechanism of these viruses in T1D development is not known. The aim of this thesis is to study various EVs that have been shown to differ in their immune phenotype, lytic ability, association with induction of islet autoantibodies, ability to replicate, cause islet disintegration and induce innate antiviral pathways in infected pancreatic cells in vitro. Furthermore, EV presence and pathogenic process in pancreatic tissue and isolated islets of T1D patients was also studied. Studies in this thesis for first time show the detection of EV RNA and protein in recent onset live T1D patients supporting the EV hypothesis in T1D development. Further all EV serotypes studied were able to replicate in islets, causing variable amount of islet disintegration ranging from extensive islet disintegration to not affecting islet morphology at all. However, one of the EV serotype replicated in only two out of seven donors infected, highlighting the importance of individual variation between donors. Further, this serotype impaired the insulin response to glucose stimulation without causing any visible islet disintegration, suggesting that this serotype might impaired the insulin response by inducing a functional block. Infection of human islets with the EV serotypes that are differentially associated with the development of islet autoantibodies showed the islet cell disintegration that is comparable with their degree of islet autoantibody seroconversion. Suggesting that the extent of the epidemic-associated islet autoantibody induction may depend on the ability of the viral serotypes to damage islet cells. Furthermore, one of the EV strains showed unique ability to infect and replicate both in endo and exocrine cells of the pancreas. EV replication in both endo and exocrine cells affected the genes involved in innate and antiviral pathways and induction of certain genes with important antiviral activity significantly varied between different donors. Suggesting that the same EV infection could result in different outcome in different individuals. Finally, we compared the results obtained by lytic and non lytic EV strains in vitro with the findings reported in fulminant and slowly progressing autoimmune T1D and found some similarities. In conclusion the results presented in this thesis further support the role of EV in T1D development and provide more insights regarding viral and host variation.  This will improve our understanding of the possible causative mechanism by EV in T1D development

Studies of Enterovirus Infection and Induction of Innate Immunity in Human Pancreatic Cells

Several epidemiological and clinical studies have indicated a possible role of Enterovirus (EV) infection in type 1 diabetes (T1D) development. However, the exact casual mechanism of these viruses in T1D development is not known. The aim of this thesis is to study various EVs that have been shown to differ in their immune phenotype, lytic ability, association with induction of islet autoantibodies, ability to replicate, cause islet disintegration and induce innate antiviral pathways in infected pancreatic cells in vitro. Furthermore, EV presence and pathogenic process in pancreatic tissue and isolated islets of T1D patients was also studied. Studies in this thesis for first time show the detection of EV RNA and protein in recent onset live T1D patients supporting the EV hypothesis in T1D development. Further all EV serotypes studied were able to replicate in islets, causing variable amount of islet disintegration ranging from extensive islet disintegration to not affecting islet morphology at all. However, one of the EV serotype replicated in only two out of seven donors infected, highlighting the importance of individual variation between donors. Further, this serotype impaired the insulin response to glucose stimulation without causing any visible islet disintegration, suggesting that this serotype might impaired the insulin response by inducing a functional block. Infection of human islets with the EV serotypes that are differentially associated with the development of islet autoantibodies showed the islet cell disintegration that is comparable with their degree of islet autoantibody seroconversion. Suggesting that the extent of the epidemic-associated islet autoantibody induction may depend on the ability of the viral serotypes to damage islet cells. Furthermore, one of the EV strains showed unique ability to infect and replicate both in endo and exocrine cells of the pancreas. EV replication in both endo and exocrine cells affected the genes involved in innate and antiviral pathways and induction of certain genes with important antiviral activity significantly varied between different donors. Suggesting that the same EV infection could result in different outcome in different individuals. Finally, we compared the results obtained by lytic and non lytic EV strains in vitro with the findings reported in fulminant and slowly progressing autoimmune T1D and found some similarities. In conclusion the results presented in this thesis further support the role of EV in T1D development and provide more insights regarding viral and host variation.  This will improve our understanding of the possible causative mechanism by EV in T1D development

Echovirus 6 infects human exocrine and endocrine pancreatic cells and induces pro-inflammatory innate immune response

Human enteroviruses (HEV), especially coxsackievirus serotype B (CVB) and echovirus (E), have been associated with diseases of both the exocrine and endocrine pancreas, but so far evidence on HEV infection in human pancreas has been reported only in islets and ductal cells. This study aimed to investigate the capability of echovirus strains to infect human exocrine and endocrine pancreatic cells. Infection of explanted human islets and exocrine cells with seven field strains of E6 caused cytopathic effect, virus titer increase and production of HEV protein VP1 in both cell types. Virus particles were found in islets and acinar cells infected with E6. No cytopathic effect or infectious progeny production was observed in exocrine cells exposed to the beta cell-tropic strains of E16 and E30. Endocrine cells responded to E6, E16 and E30 by upregulating the transcription of interferon-induced with helicase C domain 1 (IF1H1), 2ʹ-5ʹ-oligoadenylate synthetase 1 (OAS1), interferon-β (IFN-β), chemokine (C-X-C motif) ligand 10 (CXCL10) and chemokine (C-C motif) ligand 5 (CCL5). Echovirus 6, but not E16 or E30, led to increased transcription of these genes in exocrine cells. These data demonstrate for the first time that human exocrine cells represent a target for E6 infection and suggest that certain HEV serotypes can replicate in human pancreatic exocrine cells, while the pancreatic endocrine cells are permissive to a wider range of HEV

Dendrograms showing phylogenetic relationships between sequenced E30 isolated during the Cuban epidemic of aseptic meningitis in 2001 and the E30 isolate of the GenBank sequence database.

<p>The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Strains of E30 known to be highly destructive of primary human insulin producing beta cells are shown in the tree by small black triangles.</p

Viral titers of the clinical strains of E16, E30 strains and E4 in the culture medium of infected primary human islets during 3 days post-infection.

<p>Aliquots of the culture medium were withdrawn day 0 and day 3. Virus titers were obtained using the cell culture infectious dose 50 (CCID50) titration methods. The results are shown as the means ± SD from experiments performed in triplicate.</p

Innate immunity gene expression in primary human islets cultured after three days of infection with clinical strains of E4, E16 and E30.

<p>Gene expression levels are presented as mRNA expression relative to expression of the housekeeping gene 18(2^_dct). Data are presented as means ± SD and were based on observations from at least three donors. ^†<i>P</i><0.05, between all groups (Islets infected with E4, E16, E30 and uninfected controls). *<i>P</i><0.05, Islets infected with E16 and E30 were compared to E4-inoculated islets and the uninfected control.</p

Virus-induced cytopathic effect in primary human pancreatic islets cells.

<p>A. Uninfected islet. B. Islets infected with the E4 isolate 5 days post infection. C. Islets infected with E16 isolates 3 days post infection. D. Islets infected with E30 isolates 3 days post infection. The figure is representative of seven islet donors.</p