Contributions
Abstract: EP1455
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Objective: Adenosine 2a receptors (A2AR) are ubiquitous receptors in the human body. Their activation and inactivation readily regulates inflammation within the brain and the peripheral systems. A2AR antagonism by caffeine has been shown to ameliorate symptoms of EAE, an animal model of MS. Our aim was to study the effect of adenosine receptor antagonist KW6002 on neuroinflammation in an acute EAE model.
Methods: Ten 5-week-old female SJL/JCrHsd mice were immunized with PLP139-151/CFA followed by injection of PTX (kit by Hooke Laboratories). Mice were allocated to two treatment groups. First group (n=5) received daily injections of KW6002 (diluted into 5% DMSO, 5% TWEEN 80, in 0.9% NaCl) i.p. from day 5 post induction for three days at dose 3mg/kg, then for 10 days at 1,5mg/kg i.p. Sham-treated group (n=5) was given daily injections of vehicle. Scoring (0-5, 0=no symptoms, 5=moribund) was done daily. On day 21 post induction mice were euthanized and their brains and spinal cords were collected. Spinal cords were frozen, cut and stained with toluidine blue to visualize any infiltrates. The brains were used for cell sorting by FACS to isolate microglia, macrophages and T cells. RNA was extracted from the sorted cell populations with miRNeasy Micro Kit (Qiagen) and quantitative real‐time PCR (qPCR) was performed for the A2AR gene.
Results: A2AR antagonism led to earlier and more severe acute EAE. EAE disease scores were significantly greater in the KW6002-treated group on days 11 (p=0.007) and 12 (p=0.014) post induction compared to sham-treated group. T cells and macrophages retrieved from the EAE brains expressed the A2AR, but negligible A2AR expression was found on microglia. Both treatment groups had similar levels of inflammatory cell infiltrates in their spinal cords.
Conclusions: A2AR antagonism by KW6002 led to more severe disease in an acute model of EAE. This is likely due to an immunostimulatory effect on CNS-infiltrating peripheral immune cells expressing the A2AR, as brain-resident microglial cells did not express the A2AR.
Disclosure: Marjo Nylund: nothing to disclose
Anna Vuorimaa: nothing to disclose
Henrik Paavilainen: nothing to disclose
Agnieszka Wlodarczyk: nothing to disclose. AW has received funding from Lundbeck Foundation, Danish Multiple Sclerosis Society, Independent Research Fund Denmark
Katrine Jensen: nothing to disclose
Veijo Hukkanen: nothing to disclose. VH has received funding from the Academy of Finland
Bente Finsen: nothing to disclose. BF has received funding from the Danish Multiple Sclerosis Society, the Danish Research Agency, Alzheimerforskningsfonden, the Lundbeckfoundation.
Trevor Owens:nothing to disclose. AW has received funding from Lundbeck Foundation, Danish Multiple Sclerosis Society, Independent Research Fund Denmark
Laura Airas: nothing to disclose. LA has received honoraria from Biogen, F. Hoffmann-La Roche Ltd, Genzyme, Merck Serono and Teva, and institutional research grant support from Biogen, Genzyme, Merck Serono and Novartis.
Abstract: EP1455
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Experimental models
Objective: Adenosine 2a receptors (A2AR) are ubiquitous receptors in the human body. Their activation and inactivation readily regulates inflammation within the brain and the peripheral systems. A2AR antagonism by caffeine has been shown to ameliorate symptoms of EAE, an animal model of MS. Our aim was to study the effect of adenosine receptor antagonist KW6002 on neuroinflammation in an acute EAE model.
Methods: Ten 5-week-old female SJL/JCrHsd mice were immunized with PLP139-151/CFA followed by injection of PTX (kit by Hooke Laboratories). Mice were allocated to two treatment groups. First group (n=5) received daily injections of KW6002 (diluted into 5% DMSO, 5% TWEEN 80, in 0.9% NaCl) i.p. from day 5 post induction for three days at dose 3mg/kg, then for 10 days at 1,5mg/kg i.p. Sham-treated group (n=5) was given daily injections of vehicle. Scoring (0-5, 0=no symptoms, 5=moribund) was done daily. On day 21 post induction mice were euthanized and their brains and spinal cords were collected. Spinal cords were frozen, cut and stained with toluidine blue to visualize any infiltrates. The brains were used for cell sorting by FACS to isolate microglia, macrophages and T cells. RNA was extracted from the sorted cell populations with miRNeasy Micro Kit (Qiagen) and quantitative real‐time PCR (qPCR) was performed for the A2AR gene.
Results: A2AR antagonism led to earlier and more severe acute EAE. EAE disease scores were significantly greater in the KW6002-treated group on days 11 (p=0.007) and 12 (p=0.014) post induction compared to sham-treated group. T cells and macrophages retrieved from the EAE brains expressed the A2AR, but negligible A2AR expression was found on microglia. Both treatment groups had similar levels of inflammatory cell infiltrates in their spinal cords.
Conclusions: A2AR antagonism by KW6002 led to more severe disease in an acute model of EAE. This is likely due to an immunostimulatory effect on CNS-infiltrating peripheral immune cells expressing the A2AR, as brain-resident microglial cells did not express the A2AR.
Disclosure: Marjo Nylund: nothing to disclose
Anna Vuorimaa: nothing to disclose
Henrik Paavilainen: nothing to disclose
Agnieszka Wlodarczyk: nothing to disclose. AW has received funding from Lundbeck Foundation, Danish Multiple Sclerosis Society, Independent Research Fund Denmark
Katrine Jensen: nothing to disclose
Veijo Hukkanen: nothing to disclose. VH has received funding from the Academy of Finland
Bente Finsen: nothing to disclose. BF has received funding from the Danish Multiple Sclerosis Society, the Danish Research Agency, Alzheimerforskningsfonden, the Lundbeckfoundation.
Trevor Owens:nothing to disclose. AW has received funding from Lundbeck Foundation, Danish Multiple Sclerosis Society, Independent Research Fund Denmark
Laura Airas: nothing to disclose. LA has received honoraria from Biogen, F. Hoffmann-La Roche Ltd, Genzyme, Merck Serono and Teva, and institutional research grant support from Biogen, Genzyme, Merck Serono and Novartis.