PMC

TLR regulation during autoimmune CNS disease. Expression of TLRs and MyD88 in the CNS of EAE-diseased mice. TLR1–9 and MyD88 mRNA were detected in spinal cord tissue samples by using real-time PCR at different stages of disease. Immunized, nonsick animals (score 0) are indicated in white, slightly diseased mice (score 0.5–1.5) in black, and mice with severe EAE (score > 2.5) in gray. Data are expressed as ratio of induced TLR to endogenous GAPDH and expressed as SEM.

Encephalitogenic T cells are less effective at inducing EAE when transferred into MyD88- or TLR9-deficient hosts. Adoptive transfer of MOG-reactive T cells (25 × 10⁶ cells) into MyD88^–/– or TLR9^–/– but not TLR2^–/– hosts results in milder disease. Data shown are from 1 representative experiment of at least 2 individual experiments with at least 5 mice per group (means). Asterisks indicate statistical significance. P < 0.05.

Nonhematopoietic TLR signaling is involved in EAE disease. (A–C) Active EAE in MyD88 (A and B) and TLR9 (C) BM chimeric mice. After BM reconstitution, mice were allowed to recover for 6–8 weeks, then immunized with MOG_35–55 in CFA as described and scored for disease (means). Statistically significant data points are marked with asterisks. P < 0.05. Results are representative of 2 independent experiments. (B) Quantification of mononuclear infiltrates and axonal damage in MyD88 chimeric mice. For each genotype, at least 4 diseased spinal cords were used for quantification (means ± SD). Arrows indicate direction of bone marrow reconstitution.

Diminished CNS inflammation and axonal damage in MyD88- and TLR9-deficient mice. (A and B) Immunohistochemistry from WT and TLR2^–/–, TLR9^–/–, and MyD88^–/– mice. In all cases, animals with the highest clinical signs were taken either at peak of disease (day 20, A) or at the end of the experiment (day 35, B). Mac-3 staining in total spinal cord sections revealed similar strong macrophage infiltration in WT, TLR2^–/–, and TLR9^–/– mice at day 20 (A) whereas at later time points, TLR9^–/– mice revealed smaller infiltrates and MyD88^–/– mice revealed no infiltrates (B). Higher magnifications show CD3-positive lymphocytes, macrophages (Mac-3), regions of demyelination (luxol fast blue, LFB), and APP deposits representing axonal damage (APP). Scale bars: 500 μm (first column); 30 μm (second column).

TLR signaling is critical for the induction of active MOG-EAE in C57BL/6 mice and the disease-specific Th1 response. (A) EAE was induced by active immunization of WT (diamonds), TLR2^–/– (squares), TLR9^–/– (triangles), and MyD88-deficient (circles) mice. Each data point represents the mean of 6 or more animals. Statistically significant data points are marked with asterisks (P < 0.05). One representative set of experiments out of 3 is shown. (B) TLR9 engagement by selective EAE immunization components in vitro. Human TLR9 was ectopically expressed in HEK293 cells. Cells were stimulated with either 5 μl M. tuberculosis cell wall components (H37RA, 50 μg/ml), 20 μl of MOG_35–55 peptide (4 μg/μl) in sterile PBS, 20 μl PTX (1.25 ng/μl) in endotoxin-free water, or TLR-specific stimuli (2 μM oligodeoxynucleotide 2006). Luciferase activity in cellular lysates was measured, normalized to β-galactosidase activity, and related to activity in unstimulated cells in each group of vector/TLR transfectants. The empty vector is indicated by white bars and TLR9 by black. The results are representative of more than 3 independent experiments. (C and D) MyD88^–/– but not TLR9^–/– mice failed to generate Th1 immune responses. Recall responses of LN cells to either MOG_35–55 peptide or KLH were measured by IFN-γ ELISA (C) and [H]thymidine-uptake (D). Data shown are representative of at least 2 independent experiments.