Using NOD and Toll-Like Receptor 3 (TLR3) Knockout Mice in Type 1 Diabetes Research

1. Using NOD and Toll-Like Receptor 3 (TLR3) Knockout Mice in Type 1 Diabetes Research Tiffany Osemwengie

2. 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

3. Symptoms • Increased thirst • Frequent urination • Extreme hunger • Weight loss • Fatigue • Blurred vision

4. 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

5. Treatment • Fatal if left untreated • Insulin Injections • Exercising regularly and maintaining a healthy weight • Eating healthy foods • Monitoring blood sugar

6. Type-1 Diabetes Research Mouse pancreatic islet visualized using immunofluorescent microscopy

7. 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

8. 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

9. 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

10. 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

11. Toll-like Receptors

12. ZeynepDogusan 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

13. 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

14. ZeynepDogusan 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 PICexsynthetic dsRNA was shown to significantly accelerate the development of diabetes in the mice.

15. ZeynepDogusan 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

16. References • 1. ZeynepDogusan, MónicaGarcía, 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. • 2. RasschaertJ, 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 • 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