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. Author manuscript; available in PMC: 2017 Jul 10.
Published in final edited form as: J Immunol. 2014 Jul 11;193(4):1911–1919. doi: 10.4049/jimmunol.1302392

Figure 3. TRIL modulates TLR4 and TLR3 mediated in the primary murine mixed glial cells and astrocytes.

Figure 3

A, RT-PCR analysis of Tril expression in cultured microglia, astrocytes and neurons cells populations, mRNA levels of Tril were normalized to beta-Actin and expressed in arbitrary units (A.U). B, Expression of Tril at the basal level and following 5 h of stimulation with LPS (100ng/ml) or Poly(I:C) (25μg/ml) in murine mixed glial cells derived from wild type (WT) and TRIL-deficient (Tril-/-) mice, mRNA levels of Tril were normalized to beta-Actin and expressed in arbitrary units (A.U). B (histogram) FACS analysis of microglia and astrocytes composition within primary mixed glial cells. Isolated primary mixed glial cells were stained using markers specific for astrocytes (GLAST-APC; Miltenyi Biotech), microglia (Cd11b-PE; eBioscience) and neurons (β-3-Tubulin; Biolegend, not shown), graph demonstrates two main populations of cells among mixed glial cells; astrocytes and microglia, data are representative of three individual experiments. C, Gene expression analysis of primary murine mixed glial cells derived from WT and TRIL-deficient mice (Tril-/-) untreated or following 5 h of stimulation with LPS (100ng/ml) or Poly(I:C) (25μg/ml). Gene expression profiles are displayed as a heat map (log10 transformed) with hierarchical clustering indicated by dendrogram. Upregulated genes are shown in red, downregulated genes are represented in green. D-G, ELISA for IL6 (D), CCL5 (E), TNFα (F), and IFNβ (G), production in primary murine mixed glial cells derived from WT and Tril-/- mice, untreated or stimulated for 24 h with LPS (10 and 100ng/ml), Poly(I:C) (25 and 50μg/ml), Pam3CSK4 (100nM) or R848 (1μg/ml). Data are represented as the mean ± SD of one experiment representative of three independent experiments, all carried out in triplicates. ***, p<0.001, **, p<0.01, *, p<0.05, H and I, ELISA for IL6 (H) and CCL5 (I), production in the purified primary astrocyte population isolated from primary murine mixed glial cells derived from WT and TRIL-deficient mice (Tril-/-), untreated or stimulated for 24 h with LPS (100ng/ml) or Poly(I:C) (25μg/ml). Data are represented as the mean ± SEM of two independent experiments, a total of three mice were pulled in each of three experiments carried out, ***, p<0.001, **, p<0.01, *, p<0.05. J and K, ELISA for IL6 (J) and CCL5 (K), production examined in immortalized migroglia cells derived from WT and Tril-/- mice, untreated or stimulated for 24 h with LPS (100ng/ml) or Poly(I:C) (25μg/ml). Data are represented as the mean ± SEM of three independent experiments all carried out in triplicates.