Transcript Using NOD and Toll-Like Receptor 3 (TLR3) Knockout Mice

Using NOD and Toll-Like Receptor 3
(TLR3) Knockout Mice in Type 1
Diabetes Research
Tiffany Osemwengie
General Type-1 Diabetes
Information
• Type 1 diabetes mellitus is a chronic autoimmune
disease that results in the destruction of the insulinproducing cells in the pancreas.
• The insulin-producing cells are called beta cells
• The disease often results in complete loss of insulin
production in the pancreas
• Insulin is an essential hormone that is necessary to
allow glucose to enter cells to produce energy
• Also known as insulin-dependent diabetes and
juvenile diabetes
Symptoms
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Increased thirst
Frequent urination
Extreme hunger
Weight loss
Fatigue
Blurred vision
Complications of Type-1
Diabetes
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Cardiovascular Disease
Nerve damage (neuropathy)
Kidney damage
Eye damage (cataracts and blindness)
Foot damage
Skin and mouth conditions
Osteoporosis
Pregnancy complications
Hearing problems
Treatment
• Fatal if left untreated
• Insulin Injections
• Exercising regularly and maintaining a healthy
weight
• Eating healthy foods
• Monitoring blood sugar
Type-1 Diabetes Research
Mouse pancreatic islet visualized using immunofluorescent microscopy
NOD Mice
• The non-obese diabetic (NOD) mouse
• Widely used model of type-1 diabetes mellitus
• NOD mice spontaneously develop autoimmune
insulin-dependent diabetes around the age of 15
weeks
• The strain was first developed at the Shionogi
Research Laboratories in Aburahi, Japan by Makino
and colleagues in 1980
• They were developed by selecting and inbreeding
cataract-prone mice strains
NOD Mice
• The NOD mice were found to have a mutation in
exon 2 of the CTLA-4 gene
• CTLA-4 plays an important role in regulating the Tcell immune response
• It is believed that the loss of function in the CTLA-4
gene causes auto-reactive T-cells to attack insulin
producing cells, which leads to the development of
diabetes in the NOD mice
Toll-Like Receptors
• Toll-like receptors (TLRs) are cellular sensors that
detect pathogens by recognizing specific
pathogen-associated molecular patterns (PAMPs)
• Found on the surface of macrophages, dendritic
cells, and NK cells
• Can also be found in the membranes of
endosomes
• TLRs were also found to be expressed in human and
rodent pancreatic islets
• Important role in innate immune responses
• First line of defense against invading pathogens
Toll-like Receptors
• There are 10 different expressed TLR genes in
humans, and 13 in mice
• Each type of TLR recognizes a distinct set of PAMPs
• When a TLR recognizes a PAMP, a signaling process
occurs that leads to the production of cytokines
and chemokines
Toll-like Receptors
Zeynep Dogusan et al.
• It had been known previously that viral infections
could contribute to the pathogenesis of type-1
diabetes
• Viral products, especially double-stranded RNA
(dsRNA), affected pancreatic β-cell survival and
triggered autoimmunity
• The mechanism behind the induced β-cell death
was unknown
• The researchers believed that it may have
something to do with the TLR3 signaling pathway
because TLR3 recognizes viral dsRNA
TLR3 -/- Knockout Mutants
• The gene for toll-like receptor 3 in mice is located
on chromosome 8
• The gene was disrupted by targeted knockout
mutation
• Exon 1 was replaced through homologous
recombination
• Northern blot analysis detected a truncated
transcript in the knockout mutants
• The mutant transcript does not encode a functional
protein
Zeynep Dogusan et al.
• Wild-type and TLR3 -/- knockout mice were used to
determine if the TLR3 signaling pathway played a
significant role in the development of the
autoimmune condition.
• The pancreatic islets from the mice were isolated
and incubated with PICex which is a synthetic
dsRNA
• In another separate in-vivo study using NOD mice,
the PICex synthetic dsRNA was shown to
significantly accelerate the development of
diabetes in the mice.
Zeynep Dogusan et al.
• The wild-type mice had a much higher percentage
of beta cell apoptosis than the TLR3 -/- knockout
mice
• Indication that the detrimental effects of dsRNA on
pancreatic β-cells were mediated by the TLR3
signaling pathway
• Suppression of the TLR3 signaling pathway protects
β-cells against dsRNA-induced apoptosis
References
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1.
Zeynep Dogusan, Mónica García, Daisy Flamez, Lena
Alexopoulou, Michel Goldman, Conny, Gysemans, Chantal
Mathieu, Claude Libert, Decio L. Eizirik, and Joanne
Rasschaert.Double-Stranded RNA Induces Pancreatic β-Cell
Apoptosis by Activation of the Toll-Like Receptor 3 and Interferon
Regulatory Factor 3 Pathways Diabetes 2008 57: 1236-1245.
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Rasschaert J, Ladriere L, Urbain M, Dogusan Z, Katabua B,
Sato S, Akira S, Gysemans C, Mathieu C, Eizirik, DL: Toll-like
receptor 3 and STAT-1 contribute to double-stranded RNA+
interferon-gamma-induced apoptosis in primary pancreatic
beta-cells. J Biol Chem280 :33984 –33991,2005
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3.
Wen L, Peng J, Li Z, Wong, FS: The effect of innate immunity
on autoimmune diabetes and the expression of toll-like receptors
on pancreatic islets. Journal of Immunology 172 :3173 –3180,2004