Analysis of the Neuroinflammatory Response to TLR7 Stimulation in the Brain: Comparison of Multiple TLR7 and/or TLR8 Agonists

Abstract

Activation of astrocytes and microglia and the production of proinflammatory cytokines and chemokines are often associated with virus infection in the CNS as well as a number of neurological diseases of unknown etiology. These inflammatory responses may be initiated by recognition of pathogen-associated molecular patterns (PAMPs) that stimulate TLRs. TLR7 and TLR8 were identified as eliciting antiviral effects when stimulated by viral ssRNA. In the present study, we examined the potential of TLR7 and/or TLR8 agonists to induce glial activation and neuroinflammation in the CNS by intracerebroventricular inoculation of TLR7 and/or TLR8 agonists in newborn mice. The TLR7 agonist imiquimod induced astrocyte activation and up-regulation of proinflammatory cytokines and chemokines, including IFN-β, TNF, CCL2, and CXCL10. However, these responses were only of short duration when compared with responses induced by the TLR4 agonist LPS. Interestingly, some of the TLR7 and/or TLR8 agonists differed in their ability to activate glial cells as evidenced by their ability to induce cytokine and chemokine expression both in vivo and in vitro. Thus, TLR7 stimulation can induce neuroinflammatory responses in the brain, but individual TLR7 agonists may differ in their ability to stimulate cells of the CNS.

TLR7 and TLR9 Trigger Distinct Neuroinflammatory Responses in the CNS

“…19 In brief, 2-day-old mice were anesthetized by hypothermia, and 10 L of the appropriate TLR agonist in PBS/ 0.2% trypan blue was inoculated into the lateral ventricles (5 L per ventricle) using a 33-gauge needle and a Hamilton syringe. PBS with 0.2% trypan blue was used for mock ICV inoculations.…”

“…The primers to detect Ccl2, Cd3e, Cxcl10, F4/80, Gapdh, Gfap, Ifnb1, and Tnf cDNA were described previously. 19 Other primers were as follows: Il6 (5=-CCGGAGAGGAGACTTCACAG-3= forward and 5=-TC-CACGATTTCCCAGAGAAC-3= reverse), Il12 (5=-CCTGAAGT-GTGAAGCACCAA-3= forward and 5=-TCAGGGGAACTGC-TACTGCT-3= reverse), Irf7 (5=-CCAGTTGATCCGCATAA-GGT-3= forward and 5=-AGCATTGCTGAGGCTCACTT-3= reverse), MyD88 (5=-CATGGTGGTGGTTGTTTCTG-3= forward and 5=-CTGTTGGACACCTGGAGACA-3= reverse), Tlr7 (5=-GGCATTCCCACTAACACCAC-3= forward and 5=-TTGGAC-CCCAGTAGAACAGG-3= reverse), Tlr9 (5=-ACTTCGTCCAC-CTGTCCAAC-3= and 5=-TCATGTGGCAAGAGAAGTGC-3= reverse), and Ttr (5=-GCTTCCCTTCGACTCTTCCT-3= forward and 5=-GCATCCAGGACTTTGACCAT-3= reverse). All the primers were used at a concentration of 0.5 mol/L.…”

“…19 In brief, brain tissues were homogenized using Kontes disposable pellet pestles (Fisher Scientific, Hampton, NH) in 200 L of Bio-Plex cell lysis solution (Bio-Rad Laboratories) containing complete Mini protease inhibitors (Roche Applied Science, Indianapolis, IN) and 2 mmol/L phenylmethylsulfonyl fluoride. Final volumes were adjusted to 300 mg/mL of tissue with lysis buffer, and cellular debris was removed by centrifugation at 4500 ϫ g for 15 minutes at 4°C.…”

“…19 In brief, the tissue sections were fixed in 4% paraformaldehyde, and proteins were denatured in 200 mmol/L HCl followed by treatment with 5 g/mL of proteinase K. The sections were incubated overnight with digoxigenin-labeled RNA antisense or sense probes. Tissue sections were treated with RNase A and then were incubated with alkaline phosphatase antidigoxigenin antibody (Roche Diagnostics) for 60 minutes.…”

“…rebroventricular (ICV) inoculation of TLR9 agonists was lethal in newborn mice, but ICV inoculation of TLR7 agonists was not. [17][18][19] Understanding differences in the neuroinflammatory capabilities of TLR7 and TLR9 agonists will be important for using the agonists in the treatment of neurologic diseases, including neurovirulent viral infections. This is particularly true in the developing brain, where viral infection can lead to severe neurologic damage or death.…”