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 insulin- producing 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 Increased thirst Frequent urination Extreme hunger Weight loss Fatigue Blurred vision

Complications of Type-1 Diabetes 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 T- cell 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 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 : 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, 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