Apigenin and Luteolin Modulate Microglial Activation
Background. It is well known that most neurodegenerative diseases are associated with microglia-mediated inflammation. Our previous research demonstrates that the CD40 signaling is critically involved in microglia-related immune responses in the brain. For example, it is well known that the activation of the signal transducer and activator of transcription (STAT) signaling pathway plays a central role in interferon-gamma (IFN-γ)-induced microglial CD40 expression. We and others have previously reported that microglial CD40 expression is significantly induced by IFN-γ and amyloid-β (Aβ) peptide. Recent studies have shown that certain flavonoids possess anti-inflammatory and neuroprotective properties distinct from their well-known anti-oxidant effects. In particular, flavonoids, apigenin and luteolin have been found to be effective CD40 immunomodulators.
Methods. Cultured microglia, both N9 and primary derived lines, were treated with flavonoids in the presence of IFN-γ and/or CD40 ligation to assess any anti-inflammatory effects and/or mechanisms. CD40 expression on microglia was analyzed by fluorescence activated cell sorting (FACS). Anti-inflammatory effects and mechanisms were confirmed by ELISA for interlekin-6 (IL-6) and TNF-α, lactate dehydrogenase (LDH) assay, and STAT1 Western blotting.
Results. Apigenin and luteolin concentration-dependently suppressed IFN-γ-induced CD40 expression. Apigenin and luteolin also suppressed microglial TNF-α and IL-6 production stimulated by IFN-gamma challenge in the presence of CD40 ligation. In addition, apigenin and luteolin markedly inhibited IFN-γ-induced phosphorylation of STAT1 with little impact on cell survival.
Conclusion. Our findings provide further support for apigenin and luteolin's anti-inflammatory effects and suggest that these flavonoids may have neuroprotective/disease-modifying properties in various neurodegenerative disorders, including Alzheimer's disease (AD).
Multiple lines of evidence suggest microglia, the resident immune cells of the central nervous system (CNS), play a critical role in the etiology of various neurodegenerative diseases. Chronic activation of microglia is believed to trigger and maintain an inflammatory response, which may ultimately lead to neuronal cell death such as that observed in Alzheimer's disease (AD), HIV-dementia, Parkinson's disease, prion disease, amyotrophic lateral sclerosis, and multiple sclerosis. In fact, this chronic activation exposes the CNS to elevated levels of a wide array of potentially neurotoxic molecules including pro-inflammatory cytokines, complement proteins, proteinases, and reactive oxygen species (ROS). Conversely, an alternative view suggests that dysregulation of microglial activation may prevent appropriate immune responses necessary to respond to neuroinsults. Essential to microglial activation is the stimulatory signal from CD40 ligation. CD40 and its ligand (CD40L) are key immunoregulatory molecules that provide co-stimulatory input to cells from both the innate and adaptive arms of the immune system. The classical stimulatory signal for microglial activation is propagated by T-cell release of interferon-gamma (IFN-γ), which consequently sensitizes the microglia by upregulating the expression of various immunoregulatory molecules, including CD40, on their cell surfaces. Furthermore, it is well known that the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a central role in this IFN-γ-induced microglial CD40 expression. We have previously reported that microglial CD40 expression is significantly increased by IFN-γ in the presence of β-amyloid (Aβ) peptide via STAT1 activation. Accordingly, modulation of the JAK/STAT signaling pathway may not only prove to be an effective means for suppressing microglial-mediated inflammation but also an important target for neurodegenerative disease therapy.
While several anti-inflammatory drugs have been found to prevent microglial-mediated inflammation, their underlying mechanisms remain unclear and the search for more effective practical compounds continues. Recent research has focused on the analysis of flavonoids, which epidemiological study suggest are beneficial against the neurodegeneration and aging processes. Flavonoids, a group of phenolic phytochemicals, are common in vascular plants and are abundant in particular spices, vegetables, and fruits. They are considered important constituents in the human diet, although their daily intake varies with dietary habits. Several medicinal properties have been ascribed to flavonoids, notably anti-oxidant, anti-carcinogenic, and anti-inflammatory activity. One such flavonoid, apigenin, and its phase I metabolite, luteolin, have been found to reduce CD40 and CD40L expression on dendritic cells and basophils, respectively. Previous research has also shown apigenin's ability to inhibit pro-inflammatory cytokines production by monocytes, macrophages, and microglia and further substantiates this compound as versatile immunomodulator.
In the present study, we investigate the potential anti-inflammatory effects and mechanisms of these flavonoids, apigenin and luteolin, in cultured microglia. Our findings demonstrate that treatment of both N9 and murine-derived primary microglia cell lines with apigenin and luteolin significantly reduces CD40 expression induced by IFN-γ. This reduction is paralleled by significant decreases in the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by the microglia. Furthermore, data show that apigenin and luteolin treatments achieve these reductions through inactivation of STAT1 and suggest a mechanism whereby these compounds may prove to be an effective therapy for neurodegeneration.
Background. It is well known that most neurodegenerative diseases are associated with microglia-mediated inflammation. Our previous research demonstrates that the CD40 signaling is critically involved in microglia-related immune responses in the brain. For example, it is well known that the activation of the signal transducer and activator of transcription (STAT) signaling pathway plays a central role in interferon-gamma (IFN-γ)-induced microglial CD40 expression. We and others have previously reported that microglial CD40 expression is significantly induced by IFN-γ and amyloid-β (Aβ) peptide. Recent studies have shown that certain flavonoids possess anti-inflammatory and neuroprotective properties distinct from their well-known anti-oxidant effects. In particular, flavonoids, apigenin and luteolin have been found to be effective CD40 immunomodulators.
Methods. Cultured microglia, both N9 and primary derived lines, were treated with flavonoids in the presence of IFN-γ and/or CD40 ligation to assess any anti-inflammatory effects and/or mechanisms. CD40 expression on microglia was analyzed by fluorescence activated cell sorting (FACS). Anti-inflammatory effects and mechanisms were confirmed by ELISA for interlekin-6 (IL-6) and TNF-α, lactate dehydrogenase (LDH) assay, and STAT1 Western blotting.
Results. Apigenin and luteolin concentration-dependently suppressed IFN-γ-induced CD40 expression. Apigenin and luteolin also suppressed microglial TNF-α and IL-6 production stimulated by IFN-gamma challenge in the presence of CD40 ligation. In addition, apigenin and luteolin markedly inhibited IFN-γ-induced phosphorylation of STAT1 with little impact on cell survival.
Conclusion. Our findings provide further support for apigenin and luteolin's anti-inflammatory effects and suggest that these flavonoids may have neuroprotective/disease-modifying properties in various neurodegenerative disorders, including Alzheimer's disease (AD).
Multiple lines of evidence suggest microglia, the resident immune cells of the central nervous system (CNS), play a critical role in the etiology of various neurodegenerative diseases. Chronic activation of microglia is believed to trigger and maintain an inflammatory response, which may ultimately lead to neuronal cell death such as that observed in Alzheimer's disease (AD), HIV-dementia, Parkinson's disease, prion disease, amyotrophic lateral sclerosis, and multiple sclerosis. In fact, this chronic activation exposes the CNS to elevated levels of a wide array of potentially neurotoxic molecules including pro-inflammatory cytokines, complement proteins, proteinases, and reactive oxygen species (ROS). Conversely, an alternative view suggests that dysregulation of microglial activation may prevent appropriate immune responses necessary to respond to neuroinsults. Essential to microglial activation is the stimulatory signal from CD40 ligation. CD40 and its ligand (CD40L) are key immunoregulatory molecules that provide co-stimulatory input to cells from both the innate and adaptive arms of the immune system. The classical stimulatory signal for microglial activation is propagated by T-cell release of interferon-gamma (IFN-γ), which consequently sensitizes the microglia by upregulating the expression of various immunoregulatory molecules, including CD40, on their cell surfaces. Furthermore, it is well known that the activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays a central role in this IFN-γ-induced microglial CD40 expression. We have previously reported that microglial CD40 expression is significantly increased by IFN-γ in the presence of β-amyloid (Aβ) peptide via STAT1 activation. Accordingly, modulation of the JAK/STAT signaling pathway may not only prove to be an effective means for suppressing microglial-mediated inflammation but also an important target for neurodegenerative disease therapy.
While several anti-inflammatory drugs have been found to prevent microglial-mediated inflammation, their underlying mechanisms remain unclear and the search for more effective practical compounds continues. Recent research has focused on the analysis of flavonoids, which epidemiological study suggest are beneficial against the neurodegeneration and aging processes. Flavonoids, a group of phenolic phytochemicals, are common in vascular plants and are abundant in particular spices, vegetables, and fruits. They are considered important constituents in the human diet, although their daily intake varies with dietary habits. Several medicinal properties have been ascribed to flavonoids, notably anti-oxidant, anti-carcinogenic, and anti-inflammatory activity. One such flavonoid, apigenin, and its phase I metabolite, luteolin, have been found to reduce CD40 and CD40L expression on dendritic cells and basophils, respectively. Previous research has also shown apigenin's ability to inhibit pro-inflammatory cytokines production by monocytes, macrophages, and microglia and further substantiates this compound as versatile immunomodulator.
In the present study, we investigate the potential anti-inflammatory effects and mechanisms of these flavonoids, apigenin and luteolin, in cultured microglia. Our findings demonstrate that treatment of both N9 and murine-derived primary microglia cell lines with apigenin and luteolin significantly reduces CD40 expression induced by IFN-γ. This reduction is paralleled by significant decreases in the release of the pro-inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) by the microglia. Furthermore, data show that apigenin and luteolin treatments achieve these reductions through inactivation of STAT1 and suggest a mechanism whereby these compounds may prove to be an effective therapy for neurodegeneration.