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Echovirus infection in insulin producing beta cell lines: studies on released exosomes

Aziz, Kosrat (2015) BION01 20121
Degree Projects in Biology
Abstract
Type 1 Diabetes (T1D) is an immune-mediated disease where the pancreatic beta cells are selectively destroyed by autoreactive CD8+ T cells. However, the triggering events that eventually leads to autoreactive CD8 T cells infiltrating the pancreatic islets and, subsequently, destroying them still needs to be unveiled. Genetic and epidemiological evidence points to a substantial influence of environmental factors on diabetes development. Between the several possible candidates, human enteroviruses (HEV) have attracted the greatest attention as a putative trigger or accelerant of the disease. However, well-grounded mechanistic explanations for exactly how viruses may alter beta cells function and survival and how HEV can induce immunological... (More)
Type 1 Diabetes (T1D) is an immune-mediated disease where the pancreatic beta cells are selectively destroyed by autoreactive CD8+ T cells. However, the triggering events that eventually leads to autoreactive CD8 T cells infiltrating the pancreatic islets and, subsequently, destroying them still needs to be unveiled. Genetic and epidemiological evidence points to a substantial influence of environmental factors on diabetes development. Between the several possible candidates, human enteroviruses (HEV) have attracted the greatest attention as a putative trigger or accelerant of the disease. However, well-grounded mechanistic explanations for exactly how viruses may alter beta cells function and survival and how HEV can induce immunological response against beta cells leading to T1D are still lacking. The study aimed at elucidating whether rodent beta cells are susceptible to HEV infection and the consequence of such as infection for beta cell lysis and beta cell function. Also evaluate whether beta cells infected with Enterovirus (HEV) strains or under inflammatory stimuli will secret exosomes that can stimulate Ag-specific T-cell responses in draining lymph node T-cells. Further establish whether beta cells-derived exosomes contain viral RNA sequences.

Rodent Insulinoma INS-1, MIN6 and NIT-1cells were infected with the HEV strains E4, E16, and E30. Viral replication, degree of cytolysis and insulin release in response to high glucose was measured. Exosomes from NIT1 cells were isolated by ultracentrifugation and characterized by electron microscopy and flow cytometry. NIT 1 cells were infected with E16 or stimulated with pro-inflammatory cytokines and the secreted exosomes were used to test T cell reactivity in pancreatic lymph node from NOD/Lt mice. Total RNA from exosomes derived from NIT-1 cells infected by E16 was extracted and cDNA synthesized, whole genome amplified by PCR with primers encompasses the whole viral sequences.

Results (in the present) study show that beta cells are susceptible to become directly infected by HEV in vitro. The strain of E16 and E30 did replicate in beta cell derived lines (INS1, MIN6, and NIT1) cells and resulted in a pronounced cytopathic effect. E4 replicated in all examined insulinoma cells with no apparent cell destruction. The insulin release in response to high glucose stimulation was hampered in infected cells. NOD mice NIT 1 cells release exosomes that were visualized by electron microscopy as small vesicles, typically 40-80 nm in diameter. Flow cytometry analysis of exosomes attached to anti-CD63 latex beads revealed that they were positive for the tetraspanins CD63, CD9 or CD81, which are proteins often enriched in the exosomal membrane. Exosomes released by the NIT-1 insulinoma during inflammatory responses stimulated CD8 +T cell responses in draining lymph node from NOD mice. Exosomes from cultured NIT-1 cells infected with HEV-E16 showed no CD8+ T-cell proliferation. CD4+ T-cells failed to proliferate in response to exosomes from infected/inflamed NIT-1 cells. Notably, exosomes secreted by infected beta cells were enriched with HEV RNA.

The conclusive result of the present study consistently demonstrates the ability of HEV to infect pancreatic beta-cells and cause cell death and beta cell dysfunction. This study provides, for the first time, mechanistic insight into the role of beta cells exosomes as signaling factors between beta cells and the immune system and how they might be involved in the initiating of cellular immune responses in T1D. The presence of viral RNA in exosomes isolated from infected beta cells could be considered as a novel mechanism of exosome-mediated transmission of HEV between beta cells. (Less)
Popular Abstract (Swedish)
Virusinfektioner och Diabetes?


Diabetes kännetecknas av konstant förhöjt blodsocker. Sjukdomen ökar i lavinartade former och beskrivs som en av det moderna samhällets stora epidemier. Diabetes förekommer vanligtvis i två huvudsakliga former, typ1 och typ 2. I typ I-diabetes (T1D), som framförallt drabbar barn och ungdomar, har de insulinproducerande beta-cellerna förstörts av kroppens immunförsvar. Individer med T1D måste därför behandlas med insulin livet ut. Beta-cellerna hos individer med typ 2 är också förstörda men till mindre grad än i typ 1.

Genetiska och epidemiologiska studier har visat en stark korrelation mellan polymorfism i vissa genetiska loci såsom human leukocyte antigen (HLA) class II och cytotoxisk T lymfocyt... (More)
Virusinfektioner och Diabetes?


Diabetes kännetecknas av konstant förhöjt blodsocker. Sjukdomen ökar i lavinartade former och beskrivs som en av det moderna samhällets stora epidemier. Diabetes förekommer vanligtvis i två huvudsakliga former, typ1 och typ 2. I typ I-diabetes (T1D), som framförallt drabbar barn och ungdomar, har de insulinproducerande beta-cellerna förstörts av kroppens immunförsvar. Individer med T1D måste därför behandlas med insulin livet ut. Beta-cellerna hos individer med typ 2 är också förstörda men till mindre grad än i typ 1.

Genetiska och epidemiologiska studier har visat en stark korrelation mellan polymorfism i vissa genetiska loci såsom human leukocyte antigen (HLA) class II och cytotoxisk T lymfocyt antigen 4 (CTLA-4). Nästintill 50% av risken att drabbas av T1D är baserad på genetiska faktorer och ca 95% av alla T1D fall är bärare av HLA riskallel.

En av de yttre miljöfaktorerna som kan generera en inflammatorisk miljö är humana enterovirus (HEV) infektioner. Hur viruset aktiverar immunsystemet under dess tidiga möte är fortfarande okänt. Viruset har orsakat tre storskaliga epidemier som var associerade med utveckling av diabetes på Kuba. Virusets infektion i människan är mild och begränsad till mag- och tarmkanalen, fast vid svårare infektion kan viruset sprida sig till sekundära organ såsom hjärta, hjärna och bukspottkörteln. Viruset förstör direkt beta-cellerna och det resulterar i en pro-¬inflammatorisk aktivering av immunsystemet. Som modeller användes beta-celler från olika gnagare, råtta samt två muslinjer (varav en var ”non-obese”), INS¬1 insulinoma cell linje (fr. råtta), MIN6 insulinoma (fr. mus) och NIT¬1 insulinoma (fr. mus).

Denna studie har också undersökt om extracellulära vesiklar, exosomer, som frisläpps från många celler i människan kan ha någon betydande roll i relationen mellan virus och skadan på beta cellerna i bukspottkörteln. Studien har undersökt om beta celler infekterade med enterovirus eller under inflammatoriska stimuli utsöndrar exosomer som resulterar i stimulering av antigen¬-specifika T-¬celler. Flödescytometri och PCR användes där vi hade möjlighet att karaktärisera exosomer på ytmarkörer och med med PCR för att undersöka innehållet i exosomerna. Transmissions-elektron mikroskopi har använts för att visualisera exosomerna.

Detta arbete har visat att dessa virustyper har kapacitet att skapa en pro-inflammatorisk miljö som aktiverar autoreaktiva celler. Studien har visat att dessa virus kan använda sig av exosomer som transportkapslar för att sprida virala partiklar eller undgå upptäckt av immunsystemet.

Resultat tyder på att dessa virus typer utsätter cellerna för stress och eller funktionsnedsättning där insulinproduktionens svar på glukosstimulans är allvarligt påverkad. Studien förklarar med experiment att denna påverkan på cellerna kan vara en bakomliggande faktor till den försämrade insulinfrisättningen och riktar diskussionen mot de miljöfaktorer som påverkar individer där dessa virus cirkulerar.



Handledare: Corrado Cilio
Examensarbete för masterexamen 45hp, 2015
Biologiska institutionen, Lunds universitet
Dept. of Clinical Sciences,
Skåne University Hospital, Lund University (Less)
Please use this url to cite or link to this publication:
author
Aziz, Kosrat
supervisor
organization
course
BION01 20121
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
5276889
date added to LUP
2015-04-21 09:27:08
date last changed
2015-04-21 09:27:08
@misc{5276889,
  abstract     = {Type 1 Diabetes (T1D) is an immune-mediated disease where the pancreatic beta cells are selectively destroyed by autoreactive CD8+ T cells. However, the triggering events that eventually leads to autoreactive CD8 T cells infiltrating the pancreatic islets and, subsequently, destroying them still needs to be unveiled. Genetic and epidemiological evidence points to a substantial influence of environmental factors on diabetes development. Between the several possible candidates, human enteroviruses (HEV) have attracted the greatest attention as a putative trigger or accelerant of the disease. However, well-grounded mechanistic explanations for exactly how viruses may alter beta cells function and survival and how HEV can induce immunological response against beta cells leading to T1D are still lacking. The study aimed at elucidating whether rodent beta cells are susceptible to HEV infection and the consequence of such as infection for beta cell lysis and beta cell function. Also evaluate whether beta cells infected with Enterovirus (HEV) strains or under inflammatory stimuli will secret exosomes that can stimulate Ag-specific T-cell responses in draining lymph node T-cells. Further establish whether beta cells-derived exosomes contain viral RNA sequences. 

Rodent Insulinoma INS-1, MIN6 and NIT-1cells were infected with the HEV strains E4, E16, and E30. Viral replication, degree of cytolysis and insulin release in response to high glucose was measured. Exosomes from NIT1 cells were isolated by ultracentrifugation and characterized by electron microscopy and flow cytometry. NIT 1 cells were infected with E16 or stimulated with pro-inflammatory cytokines and the secreted exosomes were used to test T cell reactivity in pancreatic lymph node from NOD/Lt mice. Total RNA from exosomes derived from NIT-1 cells infected by E16 was extracted and cDNA synthesized, whole genome amplified by PCR with primers encompasses the whole viral sequences. 

Results (in the present) study show that beta cells are susceptible to become directly infected by HEV in vitro. The strain of E16 and E30 did replicate in beta cell derived lines (INS1, MIN6, and NIT1) cells and resulted in a pronounced cytopathic effect. E4 replicated in all examined insulinoma cells with no apparent cell destruction. The insulin release in response to high glucose stimulation was hampered in infected cells. NOD mice NIT 1 cells release exosomes that were visualized by electron microscopy as small vesicles, typically 40-80 nm in diameter. Flow cytometry analysis of exosomes attached to anti-CD63 latex beads revealed that they were positive for the tetraspanins CD63, CD9 or CD81, which are proteins often enriched in the exosomal membrane. Exosomes released by the NIT-1 insulinoma during inflammatory responses stimulated CD8 +T cell responses in draining lymph node from NOD mice. Exosomes from cultured NIT-1 cells infected with HEV-E16 showed no CD8+ T-cell proliferation. CD4+ T-cells failed to proliferate in response to exosomes from infected/inflamed NIT-1 cells. Notably, exosomes secreted by infected beta cells were enriched with HEV RNA. 

The conclusive result of the present study consistently demonstrates the ability of HEV to infect pancreatic beta-cells and cause cell death and beta cell dysfunction. This study provides, for the first time, mechanistic insight into the role of beta cells exosomes as signaling factors between beta cells and the immune system and how they might be involved in the initiating of cellular immune responses in T1D. The presence of viral RNA in exosomes isolated from infected beta cells could be considered as a novel mechanism of exosome-mediated transmission of HEV between beta cells.},
  author       = {Aziz, Kosrat},
  language     = {eng},
  note         = {Student Paper},
  title        = {Echovirus infection in insulin producing beta cell lines: studies on released exosomes},
  year         = {2015},
}