Pam3csk4

Assays were carried out with approximately 1 to 2 × 10⁴ cells/well of microglia seeded in 24-well tissue culture plates. At the time of the assay, the culture medium was removed and replaced with fresh medium without antibiotics, and the cells were pretreated with the pertinent inhibitors for 2 h prior to adding B. burgdorferi (MOI of 10:1). The microglial cells were incubated with the bacteria and inhibitors for a further 24 h, followed by collection of supernatant after centrifuging at 2,095 × g for 10 min at 4°C. Supernatants were stored at −20°C until analysis. The following inhibitors were used: SB203580 and BIRB796 (p38); U0126 (MEK1/2); SP600125 and Jun N-terminal kinase (JNK) inhibitor VIII (JNK) (all but one were from EMD Millipore, Billerica, MA; BIRB796 was obtained from Cayman Chemical Co., Ann Arbor, MI); and oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) (TLR2/4), CLI-095 (TLR4), Gefitinib (RipK2), and myeloid differentiation primary response 88 (MyD88) inhibitory peptide (InvivoGen, San Diego, CA).
Pam3CSK4 (Imgenex, San Diego, CA), LPS O55:B5 (Sigma-Aldrich, St. Louis, MO), or muramyl dipeptide (MDP, InvivoGen) were included as positive control agonists when required.

For stimulation of TLR-signalling, cells were incubated with 5 ng/ml LPS from E.coli 0111:B4 (Sigmal-Aldrich, L2630), 10 μg/ml Poly IC (Invivogen, Tlrl-pic), 100 nM Pam3CSK4 (Imgenex, Img2201), 100 nM R848 (Alexis, ALX-420-038-M005) or 100 nM ODN1668 (Invivogen, tlrl-1668) in cell culture medium for indicated pre-treatment times. After pre-treatment, cells were carefully washed with medium 1x and subsequently used for infections or other experiments.

The role of TLRs in inducing inflammation in oligodendrocytes, in response to B. burgdorferi exposure was determined by gene silencing using small interfering RNA (siRNA) technology. Transfection complexes were generated using 2 μL HiPerfect transfection reagent (Qiagen, Valencia, CA) and 12.5–25 nM siRNA (Santa Cruz Biotechnology, Dallas, TX) in experimental medium. The complexes were incubated at room temperature for 30 min, and 200 μL of the complex was added to cells, after replacing the old medium in the 6-well plates. Immediately after the addition of transfection complexes, 800 μL of experimental medium was added to prevent drying of cell layer. After a 24-h incubation at 37 °C, 5% CO₂, B. burgdorferi (MOI 10:1) was added and further incubated for 48 h, at the end of which, supernatants were collected as before and analyzed for chemokine and cytokine expression. A non-specific control siRNA was used as negative control for all experiments. Pam3CSK4 (Imgenex, San Diego, CA; TLR2), lipopolysaccharide (LPS) O55:B5 (Sigma Aldrich, St. Louis, MO; TLR4) were used as TLR-specific positive controls when required.