Anti-Neuroinflammatory Effects of the Calcium Channel Blocker Nicardipine on Microglial Cells: Implications for Neuroprotection

Background/Objective Nicardipine is a calcium channel blocker that has been widely used to control blood pressure in severe hypertension following events such as ischemic stroke, traumatic brain injury, and intracerebral hemorrhage. However, accumulating evidence suggests that inflammatory processes in the central nervous system that are mediated by microglial activation play important roles in neurodegeneration, and the effect of nicardipine on microglial activation remains unresolved. Methodology/Principal Findings In the present study, using murine BV-2 microglia, we demonstrated that nicardipine significantly inhibits microglia-related neuroinflammatory responses. Treatment with nicardipine inhibited microglial cell migration. Nicardipine also significantly inhibited LPS plus IFN-γ-induced release of nitric oxide (NO), and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, nicardipine also inhibited microglial activation by peptidoglycan, the major component of the Gram-positive bacterium cell wall. Notably, nicardipine also showed significant anti-neuroinflammatory effects on microglial activation in mice in vivo. Conclusion/Significance The present study is the first to report a novel inhibitory role of nicardipine on neuroinflammation and provides a new candidate agent for the development of therapies for inflammation-related neurodegenerative diseases

Regulatory Effects of Caffeic Acid Phenethyl Ester on Neuroinflammation in Microglial Cells

Microglial activation has been widely demonstrated to mediate inflammatory processes that are crucial in several neurodegenerative disorders. Pharmaceuticals that can deliver direct inhibitory effects on microglia are therefore considered as a potential strategy to counter balance neurodegenerative progression. Caffeic acid phenethyl ester (CAPE), a natural phenol in honeybee propolis, is known to possess antioxidant, anti-inflammatory and anti-microbial properties. Accordingly, the current study intended to probe the effects of CAPE on microglia activation by using in vitro and in vivo models. Western blot and Griess reaction assay revealed CAPE significantly inhibited the expressions of inducible nitric oxide synthase (NOS), cyclooxygenase (COX)-2 and the production of nitric oxide (NO). Administration of CAPE resulted in increased expressions of hemeoxygenase (HO)-1and erythropoietin (EPO) in microglia. The phosphorylated adenosine monophosphate-activated protein kinase (AMPK)-α was further found to regulate the anti-inflammatory effects of caffeic acid. In vivo results from immunohistochemistry along with rotarod test also revealed the anti-neuroinflammatory effects of CAPE in microglia activation. The current study has evidenced several possible molecular determinants, AMPKα, EPO, and HO-1, in mediating anti-neuroinflammatory responses in microglial cells

PP2Ac/STRN4 negatively regulates STING-type I IFN signaling in tumor-associated macrophages

Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8 + T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy

PP2Ac/STRN4 negatively regulates STING-type I IFN signaling in tumor-associated macrophages

Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy

PP2Ac/STRN4 negatively regulates STING-Type I interferon signaling in tumor associated macrophages

Stimulator of IFN genes type I (STING-Type I) IFN signaling in myeloid cells plays a critical role in effective antitumor immune responses, but STING agonists as monotherapy have shown limited efficacy in clinical trials. The mechanisms that downregulate STING signaling are not fully understood. Here, we report that protein phosphatase 2A (PP2A), with its specific B regulatory subunit Striatin 4 (STRN4), negatively regulated STING-Type I IFN in macrophages. Mice with macrophage PP2A deficiency exhibited reduced tumor progression. The tumor microenvironment showed decreased immunosuppressive and increased IFN-activated macrophages and CD8+ T cells. Mechanistically, we demonstrated that Hippo kinase MST1/2 was required for STING activation. STING agonists induced dissociation of PP2A from MST1/2 in normal macrophages, but not in tumor conditioned macrophages. Furthermore, our data showed that STRN4 mediated PP2A binding to and dephosphorylation of Hippo kinase MST1/2, resulting in stabilization of YAP/TAZ to antagonize STING activation. In human patients with glioblastoma (GBM), YAP/TAZ was highly expressed in tumor-associated macrophages but not in nontumor macrophages. We also demonstrated that PP2A/STRN4 deficiency in macrophages reduced YAP/TAZ expression and sensitized tumor-conditioned macrophages to STING stimulation. In summary, we demonstrated that PP2A/STRN4-YAP/TAZ has, in our opinion, been an unappreciated mechanism that mediates immunosuppression in tumor-associated macrophages, and targeting the PP2A/STRN4-YAP/TAZ axis can sensitize tumors to immunotherapy

Nicardipine suppresses LPS/IFN-γ-induced signaling pathways.

<p>BV-2 microglial cells were pretreated with nicardipine (10 μM) for 60 min, then exposed to LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 60 min. Western blot analysis was performed on whole cell lysates, and the signal intensities were normalized to total protein expression. The results are expressed as mean ± S.E.M. from 3 to 4 independent experiments. *, <i>p</i><0.05 compared with the LPS/IFN-γ treatment alone group.</p

LPS-induced intracellular IL-6 and TNF-α expression on microglia is reduced by nicardipine.

<p>Mice received intraperitoneal injections of saline or nicardipine (5 mg/kg) for 3 consecutive days. On the third day, 2 h after injection of saline or nicardipine, mice were injected with LPS (5 mg/kg) and housed for another 24 h. (A) Representative bivariate dot plots of Percoll-isolated microglial cells stained with anti-CD11b-FITC and anti-CD45-PerCP-Cy5.5. Microglia were identified by CD11b⁺/CD45^low staining. Representative histograms of intracellular IL-6 (B) and TNF-α (C) expression in isolated microglia. Mean fluorescence intensity (M.F.I.) of intracellular IL-6 and TNF-α expressed by CD11b⁺/CD45^low microglia following experimental treatments.</p

Inhibitory effect of nicardipine on LPS/IFN-γ or peptidoglycan-stimulated nitric oxide production.

<p>BV-2 microglial cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) (A) or peptidoglycan (10 μg/ml; B) for another 24 h. The culture media were collected and analyzed by a Griess reaction. Nitric oxide production is significantly different between the LPS/IFN-γ (or peptidoglycan) treatment alone and the LPS/IFN-γ (or peptidoglycan) treatment with nicardipine groups (one-way ANOVA followed by Bonferroni's <i>post hoc</i> test). The results are expressed as mean ± S.E.M. from 3 to 4 independent experiments. *, <i>p</i><0.05 compared with the control group; #, <i>p</i><0.05 compared with the LPS/IFN-γ or peptidoglycan treatment.</p

Inhibitory effect of nicardipine on LPS/IFN-γ- or peptidoglycan-stimulated iNOS and COX-2 expressions.

<p>(A and B) BV-2 microglial cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of LPS (10 ng/ml) plus IFN-γ (10 ng/ml) for another 24 h. (C and D) Cells were pretreated with different concentrations of nicardipine (1, 3, 5, or 10 μM) for 60 min before application of peptidoglycan (10 μg/ml) for another 24 h. Western blot analysis for iNOS (A and C) and COX-2 (B and D) expression was performed on whole cell lysates. The quantitative results are shown in the bottom panels. iNOS expression was significantly different between the LPS/IFN-γ (or peptidoglycan) treated-group and the group treated LPS/IFN-γ (or peptidoglycan) with nicardipine (one-way ANOVA followed by Bonferroni's post hoc test). COX-2 expression was significantly different between the LPS/IFN-γ (or peptidoglycan) treated- group and the LPS/IFN-γ (or peptidoglycan) with nicardipine treated- group (one-way ANOVA followed by Bonferroni's <i>post hoc</i> test). The results are expressed as mean ± S.E.M. from 4 to 5 independent experiments. *, <i>p</i><0.05 compared with the LPS/IFN-γ or peptidoglycan treatment.</p