Figure 3.
VPC01091.4 and AAL-149 suppress LPS-induced inflammation in macrophages and microglia. (A) HeLa cells display minimal cytotoxicity to AAL-149 and VPC01091.4 at doses up to 25 µM (mean cytotoxicity <5% for each condition, n = 3 for each dose). P < 0.0001 for all comparisons relative to lysis control. (B) Change in IL-1β expression observed by qRT-PCR in RAW 264.7 macrophages following stimulation with LPS (1 µg/ml for 3 h). Pretreatment with ≥5 µM of VPC01091.4 or 10 µM AAL-149, but not VPC01091.2-P, significantly reduced LPS-stimulated IL-1β expression. For brevity, only statistically significant comparisons from the LPS-only treated groups are shown. Box and whisker plots display the minimum, first quartile, median, third quartile, and maximum values. (C) BMDMs from WT (Trpm7fl/fl) and TRPM7 KO (Trpm7fl/flLysM Cre) mice were isolated and differentiated for 7 d prior to use for an LPS-induced inflammation assay. Expression levels of Trpm7, IL-1β, and IL-6 as measured by qRT-PCR are displayed. Levels of TRPM7 expression are normalized to control-treated WT BMDMs, and IL-1β, and IL-6 expression levels are normalized to control-treated BMDMs for their respective genotype. Four different TRPM7 inhibitors were used: 10 µM VPC4, 10 µM AAL-149, 5 µM FTY720, or 30 μM NS8593. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparison within each genotype. (D) Alveolar macrophages isolated from WT mice were stimulated with LPS as above. Pretreatment with 10 µM VPC4 significantly suppressed the expression of IL-1β, TNFα, and CCL4. (E and F) Inflammatory cytokine expression levels as measured by qRT-PCR in cultured BV-2 microglia pretreated with vehicle, 10 µM VPC01091.4 (E), or 10 µM AAL-149 (F) before stimulation with LPS (1 µg/ml for 3 h). Unless stated otherwise, ordinary one-way ANOVA was used for analyses, and error bars represent SEM. The vehicle used for VPC4, VPC2-P, FTY720, and NS8593 was DMSO, and the vehicle for AAL-149 was 100% ethyl alcohol (<0.2% vol/vol in experimental media for each). VPC4 = VPC01091.4 and VPC2-P = VPC01091.2-P.

VPC01091.4 and AAL-149 suppress LPS-induced inflammation in macrophages and microglia. (A) HeLa cells display minimal cytotoxicity to AAL-149 and VPC01091.4 at doses up to 25 µM (mean cytotoxicity <5% for each condition, n = 3 for each dose). P < 0.0001 for all comparisons relative to lysis control. (B) Change in IL-1β expression observed by qRT-PCR in RAW 264.7 macrophages following stimulation with LPS (1 µg/ml for 3 h). Pretreatment with ≥5 µM of VPC01091.4 or 10 µM AAL-149, but not VPC01091.2-P, significantly reduced LPS-stimulated IL-1β expression. For brevity, only statistically significant comparisons from the LPS-only treated groups are shown. Box and whisker plots display the minimum, first quartile, median, third quartile, and maximum values. (C) BMDMs from WT (Trpm7fl/fl) and TRPM7 KO (Trpm7fl/flLysM Cre) mice were isolated and differentiated for 7 d prior to use for an LPS-induced inflammation assay. Expression levels of Trpm7, IL-1β, and IL-6 as measured by qRT-PCR are displayed. Levels of TRPM7 expression are normalized to control-treated WT BMDMs, and IL-1β, and IL-6 expression levels are normalized to control-treated BMDMs for their respective genotype. Four different TRPM7 inhibitors were used: 10 µM VPC4, 10 µM AAL-149, 5 µM FTY720, or 30 μM NS8593. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparison within each genotype. (D) Alveolar macrophages isolated from WT mice were stimulated with LPS as above. Pretreatment with 10 µM VPC4 significantly suppressed the expression of IL-1β, TNFα, and CCL4. (E and F) Inflammatory cytokine expression levels as measured by qRT-PCR in cultured BV-2 microglia pretreated with vehicle, 10 µM VPC01091.4 (E), or 10 µM AAL-149 (F) before stimulation with LPS (1 µg/ml for 3 h). Unless stated otherwise, ordinary one-way ANOVA was used for analyses, and error bars represent SEM. The vehicle used for VPC4, VPC2-P, FTY720, and NS8593 was DMSO, and the vehicle for AAL-149 was 100% ethyl alcohol (<0.2% vol/vol in experimental media for each). VPC4 = VPC01091.4 and VPC2-P = VPC01091.2-P.

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