Extracellular vesicles derived small non-coding RNA in type 1 diabetes

UVOD Sladkorna bolezen tipa 1 nastane kot posledica avtoimunskega uničenja celic beta Langerhansovih otočkov trebušne slinavke, ki izločajo glavni anabolni hormon inzulin. Zaradi pomanjkanja lastnega hormona inzulina so osebe s sladkorno boleznijo tipa 1 primorane doživljenjsko prejemati zdravljenje z dodajanjem eksogenih odmerkov inzulina in dosledno izvajati meritve krvnega sladkorja, s čemer lahko preprečijo oziroma odložijo nastanek zapletov sladkorne bolezni. Vzrok nastanka avtoimunosti še vedno ni pojasnjen, med drugim tudi zaradi pomanjkanja bioloških označevalcev za zgodnje odkrivanje bolezni in zaradi tkivne nedostopnosti celic beta Langerhansovih otočkov v človeškem organizmu. Zunajcelični vezikli so majhne sferične strukture, ki jih celice izločajo v zunajcelični prostor z namenom celične komunikacije in usklajenega delovanja organizma. Poleg vloge pri zagotavljanju normalnega delovanja organizma lahko zunajcelični vezikli in miRNA sodelujejo tudi pri patogenezi številnih bolezni. Z doktorskim delom smo poskušali opredeliti zunajcelične vezikle kot potencialne biološke označevalce in posrednike pri nastanku sladkorne bolezni tipa 1. METODE Prisotnost zunajceličnih veziklov v izolirani frakciji krvne plazme z verjetnim izvorom iz celic beta Langerhansovih otočkov smo dokazovali z označevanjem vzorcev s specifičnimi protitelesi in slikanjem s transmisijsko elektronsko mikroskopijo. Z metodo sekvenciranja naslednje generacije smo določali miRNA zunajceličnih veziklov izoliranih iz krvne plazme desetih posameznikov s sladkorno boleznijo tipa 1 ob izbruhu bolezni, desetih posameznikov z več kot deset let trajajočo boleznijo in desetih zdravih preiskovancev. Z namenom opredelitve miRNA zunajceličnih veziklov, ki so prisotni v krvni plazmi ob intenzivnem propadu celic beta, smo kronološko analizirali tudi vzorce zunajceličnih veziklov izoliranih iz krvne plazme dveh transplantirancev Langerhansovih otočkov. Vpliv izbranih različno izraženih miRNA smo določali z in vitro testiranjem na vzorcih polne krvi, pri čemer smo spremljali izražanje označevalca zgodnje aktivacije CD69 in citotoksičnosti CD107a celic imunskega sistema s pretočno citometrijo. S fluorescenčno označeno miRNA smo s pretočno citometrijo in fluorescenčno mikroskopijo opredeljevali kopičenje veziklov v krvnih celicah imunskega sistema. Aktivacijo krvnih celic imunskega sistema ob izpostavitvi z miRNA s sintetičnimi vezikli smo utišali z dodatkom inhibitorja endosomalnih TLR7/8 receptorjev in predpostavili potencialen model učinkovanja miRNA zunajceličnih veziklov na imunski sistem. Pri preiskovancih s sladkorno boleznijo tipa 1 smo dodatno opredelili tudi dolžine telomer, ki so tudi uravnavane z miRNA in veljajo za označevalce staranja organizma ter delovanja oksidativnega stresa. REZULTATI S transmisijsko elektronsko mikroskopijo smo dokazali prisotnost zunajceličnih veziklov v krvni plazmi, ki potencialno izvirajo iz celic beta Langerhansovih otočkov, kar nakazuje na možno komunikacijo celic beta z drugimi tkivi v človeškem telesu. S sekvenciranjem naslednje generacije smo določili različno izražene miRNA pri preiskovancih s sladkorno boleznijo tipa 1 in intenzivnemu propadu celic beta ob transplantaciji Langerhansovih otočkov. Na podlagi rezultatov primerjalnih analiz izražanja miRNA smo izbrali osem zvrsti miRNA (hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-193b-5p, hsa-miR-185-5p, hsa-miR-195-3p, hsa-miR-455-5p, hsa-miR-375-3p, hsa-miR-129-5p), ki smo jih s sintetičnimi vezikli izpostavili nativnim krvnim celicam oseb s sladkorno boleznijo tipa 1 in zdravih preiskovancev pri in vitro pogojih, kar je povzročilo aktivizacijo celic imunskega sistema. Aktivacija imunskega sistema se je odražala s povečanim izražanjem zgodnjega označevalca aktivacije CD69 in citotoksičnosti CD107a celic T CD4+ in CD8+ in naravnih celic ubijalk CD56+. Pokazali smo, da se vezikli preferenčno kopičijo v fagocitnih krvnih celicah, kjer se nahajajo TLR7 in TLR8 receptorji, ki so najverjetneje odgovorni za prepoznavanje vezikularnih miRNA. Vpletenost TLR7 in TLR8 smo dokazovali s TLR7/8 inhibitorjem klorokinom, ki je ob stimulaciji celic imunskega sistema z miRNA in sintetičnimi vezikli znižal aktivacijski odziv. Prepoznavanje človeških miRNA zunajceličnih veziklov v endosomalni poti fagocitnih celic s TLR7 in TLR8 sproži imunski odziv in aktivacijo efektorskih celic imunskega sistema. Rezultati naših raziskav so pokazali tudi razlike v izražanju miRNA, katerih vloga je že bila vpisana pri regulaciji dolžin telomer. Dolžine telomer pri preiskovancih s sladkorno boleznijo tipa 1 s slabo glikemično urejenostjo so se krajšale hitreje kot pri preiskovancih z dobro glikemično urejenostjo, k čemur so morda pripomogle tudi miRNA zunajceličnih veziklov. ZAKLJUČEK Rezultati naše raziskave nakazujejo na pomembno vlogo človeških miRNA pri regulaciji imunskega sistema in staranja organizma. miRNA zunajceličnih veziklov so lahko udeležene pri aktivaciji imunskega sistema in lahko pripomorejo k nastanku začetnega vnetja Langerhansovih otočkov. Človeške miRNA tako lahko sodelujejo pri razvoju avtoimunosti in nastanku sladkorne bolezni tipa 1. Raziskava dodatno razkriva potencialne tarče za preprečevanje in zdravljenje sladkorne bolezni tipa 1.INTRODUCTION Type 1 diabetes is caused by autoimmune destruction of Langerhans islets\u27 beta-cells, resulting in an absolute lack of one’s own insulin. Hence, individuals with type 1 diabetes require a life long intensive exogenous insulin therapy with glucose monitoring to prevent the development of diabetes-related complications, which can dramatically shorten their lifespan. The cause of type 1 diabetes is still unknown due to a lack of biomarkers and difficulties in gaining the insight into beta-cells functioning during the developmental stages of the disease. With their molecular cargo extracellular vesicles can modulate a target cell response and may affect the pathogenesis of the diseases. The extracellular vesicles containing miRNAs are often studied as disease biomarkers, but rarely as mediators of development of the disease. The role of extracellular vesicles derived miRNAs in type 1 diabetes is currently not well established. Our work aimed to evaluate the extracellular vesicles as biomarkers in type 1 diabetes and evaluate their role in the modulation of the immune system in the development of the disease. METHODS Transmission electron microscopy and vesicle labelling with specific antibodies was used to characterize the extracellular vesicles originating from insulin-producing pancreatic beta cells in blood plasma. In the next step, differentially expressed extracellular vesicles’ miRNAs were evaluated using the next-generation sequencing approach in ten individuals with type 1 diabetes at the disease onset, ten individuals with ten years duration of the disease, and ten healthy controls. To assess the extracellular vesicle miRNAs in intensive beta-cell destruction, Langerhans islet transplantation individuals’ chronological blood plasma samples were sequenced and analysed. Eight differentially expressed miRNAs in type 1 diabetes and Langerhans islets destruction were selected for the in vitro immunomodulation testing with the synthetic vesicle delivery system on the whole blood samples. Flow cytometry was applied to evaluate the immune system activation with the detection of CD107a degranulation and CD69 early activation markers on CD4+ and CD8+ T-cells and CD56+ NK cells. The vesicle miRNA accumulation in the cells of the immune system was evaluated with microscopy and flow cytometry after the whole blood cells exposure to synthetic vesicles and fluorescent labeled miRNA. The endosomal TLR7/8 inhibition in the transfection experiments was performed to evaluate the role of these receptors in the recognition of extracellular vesicle delivered miRNA in the immune system activation. Additionally, miRNAs regulate telomere lengths in the cells of a human organism and these are biomarkers of oxidative stress and organsim aging. Telomere lengths were assessed in the individuals with type 1 diabetes according to their glycemic control. RESULTS Transmission electron microscopy imaging confirmed Langerhans islets beta-cell-released extracellular vesicles in the blood plasma and potential beta-cells’ communication with other tissues. Based on the next-generation sequencing differential expression analysis results, eight significantly differentially expressed miRNA in type 1 diabetes and Langerhans transplantation individuals were selected (hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-193b-5p, hsa-miR-185-5p, hsa-miR-195-3p, hsa-miR-455-5p, hsa-miR-375-3p and hsa-miR-129-5p). The selected differentially expressed miRNA delivered with synthetic vesicles to the whole blood samples from participants with type 1 diabetes and healthy controls showed the immunomodulation activity with the overexpression of an early activation marker CD69 and a cytotoxicity marker CD107a on CD4+ and CD8+ T-cells and CD 56+ NK cells. The phagocytic cells accumulated vesicles with miRNAs in the endolysosomal pathway, where TLR7 and TLR8 were expressed. The potential involvement of the endolysosomal TLR7/8 in the activation of the immune system was shown with the endosomal TLR7/8 inhibitor chloroquine, which effectively inhibited a vesicle delivered miRNAs activation. The sequencing results also revealed differentially expressed miRNAs, whose role was reported in the telomere length regulation. The telomere length erosion in the individuals with type 1 diabetes with poor glyceamic control was accelerated compared to the good glycemic controlled individuals. SUMMARY Our results illustrate miRNAs derived from human blood plasma extracellular vesicles as modulators of the immune system in type 1 diabetes autoimmunity, providing a potentially new insight into the pathogenesis of the disease, and novel molecular targets for intervention and type 1 diabetes prevention

The importance of early genetic diagnostics of hearing loss in children

Hearing loss is one of the most common sensory deficits. It carries severe medical and social consequences, and therefore, universal newborn hearing screening was introduced at the beginning of this century. Affected patients can have hearing loss as a solitary deficit (non-syndromic hearing loss) or have other organs affected as well (syndromic hearing loss). In around 60% of cases, congenital hearing loss has a genetic etiology, where disease-causing variants can change any component of the hearing pathway. Genetic testing is usually performed by sequencing. Sanger sequencing enables analysis of the limited number of genes strictly preselected according to the clinical presentation and the prevalence among the hearing loss patients. In contrast, next-generation sequencing allows broad analysis of the numerous genes related to hearing loss, exome, or the whole genome. Identification of the genetic etiology is possible, and it makes the foundation for the genetic counselling in the family. Furthermore, it enables the identification of the comorbidities that may need a referral for specialty care, allows early treatment, helps with identification of candidates for cochlear implant, appropriate aversive/protective management, and is the foundation for the development of novel therapeutic options

Novel GRHL2 gene variant associated with hearing loss

In contrast to the recessive form, hearing loss inherited in a dominant manner is more often post-lingual and typically results in a progressive sensorineural hearing loss with variable severity and late onset. Variants in the GRHL2 gene are an extremely rare cause of dominantly inherited hearing loss. Genetic testing is a crucial part of the identification of the etiology of hearing loss in individual patients, especially when performed with next-generation sequencing, enabling simultaneous analysis of numerous genes, including those rarely associated with hearing loss. We aimed to evaluate the genetic etiology of hearing loss in a family with moderate late-onset hearing loss using next-generation sequencing and to conduct a review of reported variants in the GRHL2 gene. We identified a novel disease-causing variant in the GRHL2 gene (NM_024915: c.1510C>Tp.Arg504Ter) in both affected members of the family. They both presented with moderate late-onset hearing loss with no additional clinical characteristics. Reviewing known GRHL2 variants associated with hearing loss, we can conclude that they are more likely to be truncating variants, while the associated onset of hearing loss is variable

Association of Average Telomere Length with Body-Mass Index and Vitamin D Status in Juvenile Population with Type 1 Diabetes / Povezava Povprečnih Dolžin Telomerov Z Indeksom Telesne Teže in Vitaminom D Pri Mladostnikih S Sladkorno Boleznijo Tipa 1

Izhodišče. Sladkorna bolezen tipa 1 (SBT1) je kronična avtoimunska bolezen, pri kateri hiperglikemija ter zvišana raven oksidativnega stresa in končnih produktov glikacije skupaj z genetskimi in okoljskimi dejavniki privedeta do nastanka diabetičnih zapletov. Krajše dolžine telomerov so povezane s hiperglikemičnimi epizodami in nižjimi serumskimi vrednostmi vitamina D

Contribution of Retrotransposons to the Pathogenesis of Type 1 Diabetes and Challenges in Analysis Methods

Type 1 diabetes (T1D) is one of the most common chronic diseases of the endocrine system, associated with several life-threatening comorbidities. While the etiopathogenesis of T1D remains elusive, a combination of genetic susceptibility and environmental factors, such as microbial infections, are thought to be involved in the development of the disease. The prime model for studying the genetic component of T1D predisposition encompasses polymorphisms within the HLA (human leukocyte antigen) region responsible for the specificity of antigen presentation to lymphocytes. Apart from polymorphisms, genomic reorganization caused by repeat elements and endogenous viral elements (EVEs) might be involved in T1D predisposition. Such elements are human endogenous retroviruses (HERVs) and non-long terminal repeat (non-LTR) retrotransposons, including long and short interspersed nuclear elements (LINEs and SINEs). In line with their parasitic origin and selfish behaviour, retrotransposon-imposed gene regulation is a major source of genetic variation and instability in the human genome, and may represent the missing link between genetic susceptibility and environmental factors long thought to contribute to T1D onset. Autoreactive immune cell subtypes with differentially expressed retrotransposons can be identified with single-cell transcriptomics, and personalized assembled genomes can be constructed, which can then serve as a reference for predicting retrotransposon integration/restriction sites. Here we review what is known to date about retrotransposons, we discuss the involvement of viruses and retrotransposons in T1D predisposition, and finally we consider challenges in retrotransposons analysis methods

The Role of Epigenetic Modifications in Late Complications in Type 1 Diabetes

Type 1 diabetes is a chronic autoimmune disease in which the destruction of pancreatic β cells leads to hyperglycemia. The prevention of hyperglycemia is very important to avoid or at least postpone the development of micro- and macrovascular complications, also known as late complications. These include diabetic retinopathy, chronic renal failure, diabetic neuropathy, and cardiovascular diseases. The impact of long-term hyperglycemia has been shown to persist long after the normalization of blood glucose levels, a phenomenon known as metabolic memory. It is believed that epigenetic mechanisms such as DNA methylation, histone modifications, and microRNAs, play an important role in metabolic memory. The aim of this review is to address the impact of long-term hyperglycemia on epigenetic marks in late complications of type 1 diabetes

The Role of Epigenetic Modifications in Late Complications in Type 1 Diabetes

Type 1 diabetes is a chronic autoimmune disease in which the destruction of pancreatic β cells leads to hyperglycemia. The prevention of hyperglycemia is very important to avoid or at least postpone the development of micro- and macrovascular complications, also known as late complications. These include diabetic retinopathy, chronic renal failure, diabetic neuropathy, and cardiovascular diseases. The impact of long-term hyperglycemia has been shown to persist long after the normalization of blood glucose levels, a phenomenon known as metabolic memory. It is believed that epigenetic mechanisms such as DNA methylation, histone modifications, and microRNAs, play an important role in metabolic memory. The aim of this review is to address the impact of long-term hyperglycemia on epigenetic marks in late complications of type 1 diabetes

Contribution of Retrotransposons to the Pathogenesis of Type 1 Diabetes and Challenges in Analysis Methods

Type 1 diabetes (T1D) is one of the most common chronic diseases of the endocrine system, associated with several life-threatening comorbidities. While the etiopathogenesis of T1D remains elusive, a combination of genetic susceptibility and environmental factors, such as microbial infections, are thought to be involved in the development of the disease. The prime model for studying the genetic component of T1D predisposition encompasses polymorphisms within the HLA (human leukocyte antigen) region responsible for the specificity of antigen presentation to lymphocytes. Apart from polymorphisms, genomic reorganization caused by repeat elements and endogenous viral elements (EVEs) might be involved in T1D predisposition. Such elements are human endogenous retroviruses (HERVs) and non-long terminal repeat (non-LTR) retrotransposons, including long and short interspersed nuclear elements (LINEs and SINEs). In line with their parasitic origin and selfish behaviour, retrotransposon-imposed gene regulation is a major source of genetic variation and instability in the human genome, and may represent the missing link between genetic susceptibility and environmental factors long thought to contribute to T1D onset. Autoreactive immune cell subtypes with differentially expressed retrotransposons can be identified with single-cell transcriptomics, and personalized assembled genomes can be constructed, which can then serve as a reference for predicting retrotransposon integration/restriction sites. Here we review what is known to date about retrotransposons, we discuss the involvement of viruses and retrotransposons in T1D predisposition, and finally we consider challenges in retrotransposons analysis methods

Pathogenesis of Type 1 Diabetes: Established Facts and New Insights

Type 1 diabetes (T1D) is an autoimmune disease characterized by the T-cell-mediated destruction of insulin-producing β-cells in pancreatic islets. It generally occurs in genetically susceptible individuals, and genetics plays a major role in the development of islet autoimmunity. Furthermore, these processes are heterogeneous among individuals; hence, different endotypes have been proposed. In this review, we highlight the interplay between genetic predisposition and other non-genetic factors, such as viral infections, diet, and gut biome, which all potentially contribute to the aetiology of T1D. We also discuss a possible active role for β-cells in initiating the pathological processes. Another component in T1D predisposition is epigenetic influences, which represent a link between genetic susceptibility and environmental factors and may account for some of the disease heterogeneity. Accordingly, a shift towards personalized therapies may improve the treatment results and, therefore, result in better outcomes for individuals in the long-run. There is also a clear need for a better understanding of the preclinical phases of T1D and finding new predictive biomarkers for earlier diagnosis and therapy, with the final goal of reverting or even preventing the development of the disease