Extracellular mixed histones are neurotoxic and modulate select neuroimmune responses of glial cells.

Although histone proteins are widely known for their intranuclear functions where they organize DNA, all five histone types can also be released into the extracellular space from damaged cells. Extracellular histones can interact with pattern recognition receptors of peripheral immune cells, including toll-like receptor 4 (TLR4), causing pro-inflammatory activation, which indicates they may act as damage-associated molecular patterns (DAMPs) in peripheral tissues. Very limited information is available about functions of extracellular histones in the central nervous system (CNS). To address this knowledge gap, we applied mixed histones (MH) to cultured cells modeling neurons, microglia, and astrocytes. Microglia are the professional CNS immunocytes, while astrocytes are the main support cells for neurons. Both these cell types are critical for neuroimmune responses and their dysregulated activity contributes to neurodegenerative diseases. We measured effects of extracellular MH on cell viability and select neuroimmune functions of microglia and astrocytes. MH were toxic to cultured primary murine neurons and also reduced viability of NSC-34 murine and SH-SY5Y human neuron-like cells in TLR4-dependent manner. MH did not affect the viability of resting or immune-stimulated BV-2 murine microglia or U118 MG human astrocytic cells. When applied to BV-2 cells, MH enhanced secretion of the potential neurotoxin glutamate, but did not modulate the release of nitric oxide (NO), tumor necrosis factor-α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), or the overall cytotoxicity of lipopolysaccharide (LPS)- and/or interferon (IFN)-γ-stimulated BV-2 microglial cells towards NSC-34 neuron-like cells. We demonstrated, for the first time, that MH downregulated phagocytic activity of LPS-stimulated BV-2 microglia. However, MH also exhibited protective effect by ameliorating the cytotoxicity of LPS-stimulated U118 MG astrocytic cells towards SH-SY5Y neuron-like cells. Our data demonstrate extracellular MH could both damage neurons and alter neuroimmune functions of glial cells. These actions of MH could be targeted for treatment of neurodegenerative diseases.

Natural Compounds Oridonin and Shikonin Exhibit Potentially Beneficial Regulatory Effects on Select Functions of Microglia.

Accumulating evidence indicates that the adverse neuroimmune activation of microglia, brain immunocytes that support neurons, contributes to a range of neuroinflammatory disorders, including Alzheimer's disease. Correcting the abnormal functions of microglia is a potential therapeutic strategy for these diseases. Nucleotide-binding domain leucine-rich repeat and pyrin domain-containing receptor (NLRP) 3 inflammasomes are implicated in adverse microglial activation and their inhibitors, such as the natural compounds oridonin and shikonin, reduce microglial immune responses. We hypothesized that some of the beneficial effects of oridonin and shikonin on microglia are independent of their suppression of NLRP3 inflammasomes. Murine and human microglia-like cells were stimulated with bacterial lipopolysaccharide (LPS) only, which did not induce NLRP3 inflammasome activation or the resulting secretion of interleukin (IL)-1ß, allowing for the identification of other anti-inflammatory effects. Under these experimental conditions, both oridonin and shikonin reduced nitric oxide (NO) secretion and the cytotoxicity of BV-2 murine microglia towards HT-22 murine neuronal cells, but upregulated BV-2 cell phagocytic activity. Only oridonin inhibited the secretion of tumor necrosis factor (TNF) by stimulated BV-2 microglia, while only shikonin suppressed the respiratory burst response of human HL-60 microglia-like cells. This observed discrepancy indicates that these natural compounds may have different molecular targets in microglia. Overall, our results suggest that oridonin and shikonin should be further investigated as pharmacological agents capable of correcting dysfunctional microglia, supporting their potential use in neuroinflammatory disorders.

Pro-neuroinflammatory and neurotoxic potential of extracellular histones H1 and H3.

Histones organize DNA within cellular nuclei, but they can be released from damaged cells. In peripheral tissues extracellular histones act as damage-associated molecular patterns (DAMPs) inducing pro-inflammatory activation of immune cells. Limited studies have considered DAMP-like activity of histones in the central nervous system (CNS); therefore, we studied the effects of extracellular histones on microglia, the CNS immunocytes, and on neuronal cells. Both the linker histone H1 and the core histone H3 induced pro-inflammatory activation of microglia-like cells by upregulating their secretion of NO and cytokines, including interferon-γ-inducible protein 10 (IP-10) and tumor necrosis factor-α (TNF). The selective inhibitors MMG-11 and TAK-242 were used to demonstrate involvement of toll-like receptors (TLR) 2 and 4, respectively, in H1-induced NO secretion by BV-2 microglia. H1, but not H3, downregulated the phagocytic activity of BV-2 microglia. H1 was also directly toxic to all neuronal cell types studied. We conclude that H1, and to a lesser extent H3, when released extracellularly, have the potential to act as a CNS DAMPs. Inhibition of the DAMP-like effects of extracellular histones on microglia and their neurotoxic activity represents a potential strategy for combating neurodegenerative diseases that are characterized by the adverse activation of microglia and neuronal death.