poster: characterization studies of the mad rat: type 1 diabetes
DESCRIPTION
BRM Poster Presented at QUAD: Characterization studies of the mad rat: Type 1 DiabetesTRANSCRIPT
Characterization Studies of the Multiple Autoimmune Disease (MAD) Rat:
Type 1 Diabetes
S Duclos, B.S., M Gardner, S Champagne, B.S., RLAT, C Hogan, M.S., H Johnson, K Guberski, B.S.
Biomedical Research Models Inc., Worcester, MA
.
This research and development project was supported
in part by NIH SBIR grant R43DK085910.
Many thanks to the BRM technical staff.
Biomedical Research Models, Inc. has developed an inbred rat strain
susceptible to Multiple Autoimmune Diseases (MAD). The MAD rats were
derived from congenic LEW1.WR1 rats. During inbreeding (F55) selection
was made for autoimmune features. The goal was to develop a reliable
model to test the efficacy of treatments designed to prevent or reverse
autoimmune disease and for screening for adverse effects of these
therapies.
Immunological (TLR ligands) and viral perturbants have been identified
which increase the penetrance of disease in the several rat models of type
1 diabetes (T1D) (e.g. BBDP, BBDR and KDP rats). The MAD rat has a
normal immunological phenotype but develops spontaneous autoimmune
diabetes with a frequency of ≤ 1%.The hypothesis was made that the MAD
rat strain is genetically predisposed to the development of autoimmune
diseases that may progress to overt disease in concert with appropriate
environmental perturbants. To test this hypothesis we evaluated the
induction of T1D when treated with Toll-Like receptor (TLR) ligands. A dose
response curve was also generated for virus diabetogenicity. The age of
susceptibility was also investigated to further characterize the model and
potentially enhance future application of regimens aimed at
prevention/reversal of diabetes
Biomedical Research Models, Inc.
508-459-7544
www.brmcro.com
A panel of TLR agonists was tested for their ability to induce T1D in MAD
rats. We hypothesized that appropriate activation of innate immune cells
that initiate T1D in genetically susceptible hosts may lead directly to overt
disease. The doses were chosen based on previous studies that
demonstrated diabetogenicity of certain TLR agonists in concert with
Kilham rat virus (KRV) infection in the MHC-related rat diabetes resistant
BB rat strain (BBDR) 2,3. Standard commercially available TLR agonists
were administered at 0.5, 1, and 2 times the doses previously known to
synergize with virus infections in BBDR/Wor rats, but that are not
diabetogenic in the absence of virus. We also tested both high and low
molecular weight forms of poly I:C (TLR3 agonist). Rats were dosed 3
times weekly by IP injection beginning at 21 - 24 days of age. Compounds
were administered to 6 ♀ and 6 ♂ rats per compound or dose level, as
appropriate. Rats were treated over a 30 day period and were monitored
for diabetes from 7 to 40 days after the initiation of treatment.
In addition to screening for T1D, rats treated with TLR ligands were also
examined for evidence of inflammatory arthritis, the presence of which
would be an “off-target” effect of the induction procedure.
Diabetes can be induced in the class II MHC-identical BBDR/Wor rat
strain only until 24 - 28 days of age, after which they show resistance to
induction (unpublished data). We proposed to investigate the window of
age susceptibility in the MAD rat strain to further characterize the model
and potentially enhance future application of regimens aimed at
prevention/reversal of diabetes. We chose to use our reference
diabetogenic compound HMW poly I:C (Table 1 Group 1 and Table 3) for
these studies. Groups of 6 male and 6 female rats were administered poly
I:C (1 mg/kg) three times weekly IP until diabetes onset or until 30 days
after the initiation of treatment. Treatments were initiated at ages ranging
from 21 to 44 days.
All experimental animal procedures were carried out in accordance with
the governmental guidelines and approved by the Institutional Animal
Care and Use Committee.
• Administration of our historical standard poly I:C (primarily HWM) dose
of 1.0 mg/kg resulted in a high incidence of T1D in MAD rats in rats of
both genders (Table 1).
• The MW composition of the poly I:C material has no apparent effect on
T1D onset in this model (Table 2). It has been reported that HMW poly
I:C signals through pathways different from those activated by the low
molecular weight (LMW) form, with potential implications in autoimmune
disease (4,5). Since both HMW and LMW preparations induced
comparable frequencies of T1D at similar doses and with similar kinetics
in both genders, this data suggests that a broad spectrum of double
stranded RNA molecular species may be able to trigger autoimmune
diabetes in susceptible hosts (HMW through MDA5 signaling pathway
and LMW through RIG-I signaling pathway).
• Zymosan (Table 3) and R848 (Table 4) showed a substantial ability to
induce diabetes in this rat strain, whereas CpG (Table 5) failed to cause
glycosuria or elevated blood glucose levels in MAD rats.
• These TLR study results are presented in Table 6.
• Diabetes can be induced in the class II MHC-identical BBDR/Wor rat
strain only until 24 - 28 days of age, after which they show resistance to
induction (unpublished data). We proposed to investigate the window of
age susceptibility in the MAD rat strain to further characterize the model
and potentially enhance future application of regimens aimed at
prevention/reversal of diabetes. As shown in Table 6, induction of T1D
with standard dose poly I:C is highly efficient when treatment is started
up to 34 days of age (66 - 75%); disease penetrance then diminishes
significantly at 41 - 44 days of age (27%) (Table 7). These findings will
allow for a larger window for pretreatment with potential T1D therapies
for more flexible experimental designs in future studies.
The TLR agonists studied during this characterization are known to
synergize with KRV virus infection in the BBDR rat, but, except for poly
I:C, they do not induce diabetes or insulitis by themselves when
administered in these dose ranges in short-term dosing regimens (3
times) in BBDR rats (3,6). Furthermore, administration of these agents
to the spontaneously diabetic NOD mouse either prevents or delays the
onset of T1D (6,7). These results distinguish the MAD rat model from
the NOD mouse model. The MAD rat exhibits minimal spontaneous
disease in clean housing, but is readily induced to become diabetic with
selected purified TLR agonists. In contrast, the NOD mouse model has
a high frequency of spontaneous disease that is prevented by these
TLR agonists, as well as an extensive repertoire of other interventions
(8). The interventions proven useful in NOD mice have, to date, not
proven useful in human clinical trials (9), making the MAD rat a more
attractive competitive research tool for the evaluation of candidate
interventions.
1. Mordes JP, Poussier P, Rossini AA, Blankenhorn EP, Greiner DL:
Rat models of type 1 diabetes: Genetics, environment, and
autoimmunity. In Animal Models of Diabetes: Frontiers in
Research. 2 ed. Shafrir E, Ed. Boca Raton, CRC Press, 2007, p. 1-39
2. Mordes,JP, Guberski,DL, Leif,JH, Woda,BA, Flanagan,JF,
Greiner,DL, Kislauskis,EH, Tirabassi,RS: LEW.1WR1 rats develop
autoimmune diabetes spontaneously and in response to
environmental perturbation. Diabetes 54:2727-2733, 2005
3. Zipris,D, Lien,E, Xie,JX, Greiner,DL, Mordes,JP, Rossini,AA: TLR
activation synergizes with Kilham rat virus infection to induce
diabetes in BBDR rats. J Immunol 174:131-142, 2005
4. Kato,H, Takeuchi,O, Mikamo-Satoh,E, Hirai,R, Kawai,T,
Matsushita,K, Hiiragi,A, Dermody,TS, Fujita,T, Akira,S: Length-
dependent recognition of double-stranded ribonucleic acids by
retinoic acid-inducible gene-I and melanoma differentiation-
associated gene 5. J Exp Med 205:1601-1610, 2008
5. Pichlmair,A, Schulz,O, Tan,CP, Rehwinkel,J, Kato,H, Takeuchi,O,
Akira,S, Way,M, Schiavo,G, Reis e Sousa: Activation of MDA5
requires higher-order RNA structures generated during virus
infection\. J Virol 83:10761-10769, 2009
6. Lien,E, Zipris,D: The role of Toll-like receptor pathways in the
mechanism of type 1 diabetes. Curr Mol Med 9:52-68, 2009
7. Serreze,DV, Hamaguchi,K, Leiter,EH: Immunostimulation
circumvents diabetes in NOD/Lt mice. J Autoimmun 2:759-776,
1989
8. Atkinson,M, Leiter,EH: The NOD mouse model of insulin
dependent diabetes: As good as it gets? Nature Med 5:601-604,
1999
9. Michels,AW, Eisenbarth,GS: Immune intervention in type 1
diabetes. Semin Immunol 23:214-219, 2011
Group TLR Ligand TLR Frequency of T1D Frequency of Arthritis Gender Bias
1 Poly I:C (primarily HMW) TLR3a Positive (++++)
2 LMW Polu I:C (purified) TLR3a Positive (++++)
3 HMW Poly I:C (purified) TLR3a Positive (+++)
4 Zymosan TLR2 Positive (++)
5 R848 TLR7 Positive (++)
6 CpG oligodeoxynucleotide TLR9 Negative
None ♀ = ♂
Frequency of Diabetes in MAD Rats
Rats were treated with the reagents indicated three times weekly as described in the text. aHMW: High molecular weight poly I:C, which signals through MDA5.
LMW: Low molecular weight poly I:C, which singals via RIG-I.
Group Age at Start of Treatment Frequency (%) of Diabetes Latency To Diabetes OnsetA
1 41-44 days 3/11 (27%) 20, 22, 22 days
2 31-34 days 9/12 (75%) 13, 15, 15, 15, 18, 18, 18, 18, 18 days
3 21-24 days 8/12 (66%) 11, 13, 15, 15, 18, 18, 25, 25 days
Frequency of diabetes in MAD rats treated at different ages
ALatencies are relative to the start of poly I:C treatment on day 1.
Treatment Frequency of Diabetes % Diabetes Latency To Onset (Days)
Vehicle 0/12 0 N/A
Poly I:C 0.5 mg/kg 5/12 42 14,14,21,21,23
Poly I:C 1.0 mg/kg 11/12 92 14,14,14,14,14,14,14,18,21,21,32
Poly I:C 2.0 mg/kg 7/12 58 11,14,14,18,18,25,32
Induction of Diabetes in MAD Rats by Poly I:C
Legend: MAD rats were treated with commercial poly I:C (Sigma, St. Louis, MO) as described in the text.
Polymer length of this product is not specified, but is generally of high molecular weight.
There were no statistically significant differences between genders.
Group Treatment Frequency (% Males) Latency (Males) Frequency (% Females) Latency (Females)
1 Vehicle 0/5 0% N/A 0/5 0% N/A
2 1 mg/kg Sigma Poly I:C 3/5, 60% 16, 23, 28 0/5 0% N/A
3 2.5 mg/kg Sigma Poly I:C 2/5, 40% 12, 16, 39 1/5, 20% 19
4 5 mg/kg Sigma Poly I:C 1/5, 20% 42 2/5, 40% 12, 16
5 1 mg/kg LMW Poly I:C 2/5, 40% 12, 12 1/5, 20% 21
6 2.5 mg/kg LMW Poly I:C 2/5, 40% 19, 28 4/5, 80% 14, 14, 16, 28
7 5 mg/kg LMW Poly I:C 4/5, 80% 12, 23, 23, 40 5/5, 100% 9, 12, 12, 19, 33
8 1 mg/kg HMW Poly I:C 3/5, 60% 14, 23, 40 2/5, 40% 19, 21
9 2.5 mg/kg HMW Poly I:C 4/5, 80% 12, 14, 16, 37 5/5, 100% 12, 14, 14, 19, 37
10 5 mg/kg HMW Poly I:C 5/5, 100% 12, 14, 27, 33, 37 5/5, 100% 12, 16, 19, 23, 37
Induction of T1D in MAD Rats by Poly I:C Polymers of Differing Molecular Weight
Legend: Commercial poly I:C (Sigma) and purified poly I:C of high and low molecular weight (InVivoGen) was
given to MAD rats as described in the text. There were no statistically significant differences between genders.
Treatment Frequency of Diabetes % Diabetes Latency To Onset
Vehicle 0/12 0 N/A
Zymosan 1.0 mg/kg 5/12 42 18,20,25,25,32
Zymosan 2.0 mg/kg 9/12 75 15,20,20,20,20,25,25,29,32
Zymosan 4.0 mg/kg 10/12 83 20,20,20,20,25,25,25,25,29,29
Induction of Diabetes in MAD rats by Different Doses of the TLR2 Agonist Zymosan
Legend: MAD rats were treated with the TLR9 agonist Zymosan at the indicated doses.
There were no statistically significant differences between genders.
Treatment Frequency of Diabetes % Diabetes Latency To Onset
Vehicle 0/12 0 N/A
R848 1.0 mg/kg 3/12 25 16, 18, 28
R848 2.0 mg/kg 5/12 42 18,18,18,21,28
R848 4.0 mg/kg 1/12 8 28
Legend: Purified R848 was injected intraperitoneally into MAD rats at the
Induction of Diabetes in MAD rats by Different Doses of the TLR7 Agonist R848
indicated doses. There were no statistically significant differences between genders.
Treatment Frequency of Diabetes % Diabetes
Vehicle 0/12 0
CpG 1.0 mg/kg 0/12 0
CpG 2.0 mg/kg 0/12 0
CpG 4.0 mg/kg 0/12 0
Induction of Diabetes in MAD rats by the TLR9 Agonist CpG Oligodeoxynucleotide
Legend: MAD rats were injected with the indicated doses of the TLR9 agonist
CpG Oligodeoxynucleotide at the doses indicated. There were no statistically significant
differences between genders.