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miR-154-5p Is a Novel Endogenous Ligand for TLR7 Inducing Microglial Activation and Neuronal Injury

McGurran, Hugo, Kumbol, Victor, Krüger, Christina, Wallach, Thomas, Lehnardt, Seija·2024·Cells
DOI 10.3390/cells13050407PMC PMC10930870
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Abstract

Toll-like receptors (TLRs) are a collection of pattern recognition sensors that form a first line of defence by detecting pathogen- or damage-associated molecular patterns and initiating an inflammatory response. TLR activation in microglia, the major immune cells in the brain, can trigger the release of inflammatory molecules, which may contribute to various CNS diseases including Alzheimer’s disease. Recently, some microRNAs were shown to serve as signalling molecules for TLRs. Here, we present miR-154-5p as a novel TLR7 ligand. Exposing microglia to miR-154-5p results in cytokine release and alters expression of the TLR signalling pathway dependent on TLR7. Additionally, miR-154-5p causes neuronal injury in enriched cortical neuron cultures and additive toxicity in the presence of microglia. Finally, intrathecal injection of miR-154-5p into mice leads to neuronal injury and accumulation of microglia in the cerebral cortex dependent on TLR7 expression. In conclusion, this study establishes miR-154-5p as a direct activator of TLR7 that can cause neuroinflammation and neuronal injury, which may contribute to CNS disease.

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Figure 1 miR-154-5p activates mouse and human TLR7. Human (A,B) and murine (C,D) TLR7/8 HEK293-derived reporter and corresponding null control cell lines were stimulated with synthetic miR-154-5p (10 μg/mL) or controls for 24 h. Unstimulated, Lyovec, and control oligo (10 μg/mL) conditions served as negative controls, while TLR7 agonist Loxoribine (1 mM) and TLR7/8 agonist R848 (100 ng/mL) served as positive controls. Results are expressed as the mean ± SEM with n = 3. Significance determined using Student’s t-test compared to respective control line with same condition (*) or by Student’s t-test compared to miR-154-5p (#/n.s). (E) TNF levels in the supernatant were assessed by ELISA after 24 h stimulation of both wild-type (WT) and Tlr7−/− microglia with Lyovec, control oligo (10 μg/mL), miR-154-5p (10 μg/mL), or Loxoribine (1 mM). Significance determined by Student’s t-test relative to Tlr7−/− levels for indicated condition. (F) Dose response of WT microglia after 24 h stimulation with indicated concentration of miR-154-5p. Significance determined One-way ANOVA followed by Dunnett’s test for multiple comparison compared to unstimulated (*) or by Student’s t-test compared to miR-154-5p (n.s). (G) Representative images of WT and Tlr7−/− microglia immunostained with IBA1 antibody and treated with control oligo (10 μg/mL), miR-154-5p (10 μg/mL), Loxoribine (1 mM), or LPS (100 ng/mL, TLR4 agonist) for 24 h, demonstrating alterations in microglial morphology. Scale bar represents 50 μm. */# p < 0.05, **/## p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 1 miR-154-5p activates mouse and human TLR7. Human (A,B) and murine (C,D) TLR7/8 HEK293-derived reporter and corresponding null control cell lines were stimulated with synthetic miR-154-5p (10 μg/mL) or controls for 24 h. Unstimulated, Lyovec, and control oligo (10 μg/mL) conditions served as negative controls, while TLR7 agonist Loxoribine (1 mM) and TLR7/8 agonist R848 (100 ng/mL) served as positive controls. Results are expressed as the mean ± SEM with n = 3. Significance determined using Student’s t-test compared to respective control line with same condition (*) or by Student’s t-test compared to miR-154-5p (#/n.s). (E) TNF levels in the supernatant were assessed by ELISA after 24 h stimulation of both wild-type (WT) and Tlr7−/− microglia with Lyovec, control oligo (10 μg/mL), miR-154-5p (10 μg/mL), or Loxoribine (1 mM). Significance determined by Student’s t-test relative to Tlr7−/− levels for indicated condition. (F) Dose response of WT microglia after 24 h stimulation with indicated concentration of miR-154-5p. Significance determined One-way ANOVA followed by Dunnett’s test for multiple comparison compared to unstimulated (*) or by Student’s t-test compared to miR-154-5p (n.s). (G) Representative images of WT and Tlr7−/− microglia immunostained with IBA1 antibody and treated with control oligo (10 μg/mL), miR-154-5p (10 μg/mL), Loxoribine (1 mM), or LPS (100 ng/mL, TLR4 agonist) for 24 h, demonstrating alterations in microglial morphology. Scale bar represents 50 μm. */# p < 0.05, **/## p < 0.01, *** p < 0.001, **** p < 0.0001.CC BY · PubMed Central
Figure 2 miR-154-5p alters components of the TLR signalling pathway in microglia. WT and Tlr7−/− microglia were stimulated with miR-154-5p (10 μg/mL) for 24 h, then analysed for TLR signalling pathway components by RT-qPCR. Data is expressed as the mean fold change ± SEM compared to unstimulated control as indicated by the dashed line at y = 1.
Figure 2 miR-154-5p alters components of the TLR signalling pathway in microglia. WT and Tlr7−/− microglia were stimulated with miR-154-5p (10 μg/mL) for 24 h, then analysed for TLR signalling pathway components by RT-qPCR. Data is expressed as the mean fold change ± SEM compared to unstimulated control as indicated by the dashed line at y = 1.CC BY · PubMed Central
Figure 3 miR-154-5p causes dose- and time-dependent neuronal injury. (A,B) Primary neuron cultures were stimulated for 5 or 8 days with control oligo (10 μg/mL) as a negative control, indicated dose of miR-154-5p, or Loxoribine (1 mM) as a positive control, and cell toxicity was assessed by quantification of NeuN- or TUNEL-positivity. (C) Representative images based on percentage change to unstimulated for 8 day stimulation with control oligo (10 μg/mL), miR-154-5p (10 μg/mL) or Loxoribine (1 mM). (D) Primary Tlr7−/− neurons were stimulated for 5 or 8 days with control oligo (10 μg/mL) as a negative control, miR-154-5p (10 μg/mL), or LPS (100 ng/mL) as a positive control for microglial activation and immunostained with NeuN antibody for quantification. (E) Primary neuronal and microglial co-cultures were stimulated with indicated dose of miR-154-5p for 5 days and subsequently immunostained with NeuN antibody and quantified. (F) Representative images of co-cultures immunostained with NeuN and IBA1 antibodies after 5 days stimulation with miR-154-5p (10 μg/mL) or control oligo (10 μg/mL) and Loxoribine (1 mM) serving as controls. Scale bar represents 25 μm. Significance was determined using one-way ANOVA followed by Dunnett’s test compared to unstimulated control (*) or Control oligo (†) or by Student’s t-test comparing the highest dose miR-154-5p to positive control (n.s/#). */†/# p < 0.05, **/‡ p < 0.01, ***/†† p < 0.001, **** p < 0.0001.
Figure 3 miR-154-5p causes dose- and time-dependent neuronal injury. (A,B) Primary neuron cultures were stimulated for 5 or 8 days with control oligo (10 μg/mL) as a negative control, indicated dose of miR-154-5p, or Loxoribine (1 mM) as a positive control, and cell toxicity was assessed by quantification of NeuN- or TUNEL-positivity. (C) Representative images based on percentage change to unstimulated for 8 day stimulation with control oligo (10 μg/mL), miR-154-5p (10 μg/mL) or Loxoribine (1 mM). (D) Primary Tlr7−/− neurons were stimulated for 5 or 8 days with control oligo (10 μg/mL) as a negative control, miR-154-5p (10 μg/mL), or LPS (100 ng/mL) as a positive control for microglial activation and immunostained with NeuN antibody for quantification. (E) Primary neuronal and microglial co-cultures were stimulated with indicated dose of miR-154-5p for 5 days and subsequently immunostained with NeuN antibody and quantified. (F) Representative images of co-cultures immunostained with NeuN and IBA1 antibodies after 5 days stimulation with miR-154-5p (10 μg/mL) or control oligo (10 μg/mL) and Loxoribine (1 mM) serving as controls. Scale bar represents 25 μm. Significance was determined using one-way ANOVA followed by Dunnett’s test compared to unstimulated control (*) or Control oligo (†) or by Student’s t-test comparing the highest dose miR-154-5p to positive control (n.s/#). */†/# p < 0.05, **/‡ p < 0.01, ***/†† p < 0.001, **** p < 0.0001.CC BY · PubMed Central
Figure 4 Intrathecal injection of miR-154-5p into mice induces microglial accumulation and neuronal injury. (A) Mice were intrathecally injected with miR-154-5p (10 μg/mL) or control oligo (10 μg/mL) and sacrificed after 3 days. Sections were immunostained with IBA1 and NeuN antibodies to mark microglia and neurons, respectively. Microglia were quantified in 6 total fields of both hemispheres at 20× magnification. (B) Representative images based on average number of microglia per field. Scale bar represents 50 μm. (C) Average number of NeuN-positive cells in 6 fields of both hemispheres. (D) Representative images based on average number of NeuN-positive cells per field. Scale bar represents 50 μm. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test on all conditions to account for both treatment and genetic background. * p < 0.05, ** p < 0.01.
Figure 4 Intrathecal injection of miR-154-5p into mice induces microglial accumulation and neuronal injury. (A) Mice were intrathecally injected with miR-154-5p (10 μg/mL) or control oligo (10 μg/mL) and sacrificed after 3 days. Sections were immunostained with IBA1 and NeuN antibodies to mark microglia and neurons, respectively. Microglia were quantified in 6 total fields of both hemispheres at 20× magnification. (B) Representative images based on average number of microglia per field. Scale bar represents 50 μm. (C) Average number of NeuN-positive cells in 6 fields of both hemispheres. (D) Representative images based on average number of NeuN-positive cells per field. Scale bar represents 50 μm. Significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test on all conditions to account for both treatment and genetic background. * p < 0.05, ** p < 0.01.CC BY · PubMed Central

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Metadata

Authors
McGurran, H., Kumbol, V., Krüger, C., Wallach, T., & Lehnardt, S.
Affiliations
  • Charité—Universitätsmedizin Berlin, Einstein Center for Neurosciences Berlin, 10117 Berlin, Germany
  • Institute of Cell Biology and Neurobiology, Charité—Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität zu Berlin and Berlin Institute of Health, 10117 Berlin, Germany
Venue
Cells
Publisher
MDPI AG
Published
2024-02-26
Volume
13
Issue
5
Pages
407
ISSN
2073-4409
DOI
10.3390/cells13050407
PMC
PMC10930870
Cited by
12
References
3
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
Deutsche Forschungsgemeinschaft (SFB-TRR 167/B03, LE 2420/7-1)

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