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Fig. 2 | Molecular Neurodegeneration

Fig. 2

From: MicroRNA-100-5p and microRNA-298-5p released from apoptotic cortical neurons are endogenous Toll-like receptor 7/8 ligands that contribute to neurodegeneration

Fig. 2

Extracellular miRNAs activate human TLR7 and TLR8 expressed in HEK TLR reporter cells, as well as murine microglia and human monocytes, depending on their sequence. HEK-Blue cells co-expressing human TLR7 a or human TLR8 b and an NF-κB/AP1-inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene (a, b) were incubated with 20 μg/ml of indicated miRNAs (for miRNA concentrations given in [nm] please refer to Additional Table 2), loxoribine (a, b, 1 mM, TLR7 agonist), R848 (b, 100 ng/ml, TLR7/8 agonist), or TNF-α (a, b, 100 ng/ml, SEAP induction) for 24 h. Unstimulated HEK-Blue TLR-expressing cells and HEK-Blue Null1 or Null1-k cells served as negative control. Data are expressed as fold change of optical density of the SEAP protein normalized to unstimulated control. Data are represented as mean ± SD (n = 3). *P < 0.05; **P < 0.01; ****P < 0.0001 compared to the corresponding Null1 or Null1-k control, two-tailed Student’s t-test. (c) Microglia from C57BL/6 (wild-type, WT, n = 4) or Tlr7−/− (n = 3) mice were incubated with 5 μg/ml of indicated miRNAs for 24 h followed by TNF-α ELISA. Unstimulated cells served as negative control. LPS (100 ng/ml) and loxoribine (1 mM) served as positive control. Data are expressed as mean ± SD. *P < 0.05; ***P < 0.001; ****P < 0.0001 compared to control, Student’s t-test. (d) Macrophages differentiated from THP-1 cells were incubated with 5 μg/ml of indicated miRNAs for 24 h. Unstimulated cells served as negative control. LPS (100 ng/ml) and R848 (100 ng/ml) served as positive control. TNF-α amounts in supernatants were subsequently quantified by ELISA. All results are shown as mean ± SD with n = 6. *P < 0.05; **P < 0.01 compared to control, one-way ANOVA with Holm-Sidak’s multiple comparison test

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