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Toll-like receptor 2 (TLR2) was recently shown to contribute to secondary brain damage after intracerebral hemorrhage (ICH), although the molecular mechanisms of this contribution are elusive. In this study, we tested the hypothesis that hemin functions as a TLR2 endogenous agonist, causing proinflammatory astrocyte activation and secondary brain damage after ICH. Hemin administration to the mouse brain striatum induced ICH injury and neurological deficits, however, the brain injury volume and neurological deficits due to hemin injection were significantly reduced in TLR2 knock-out (KO) mice. Hemin administration induced neutrophil infiltration and upregulated neutrophil-attracting chemokine and proinflammatory cytokine expression in wild-type (WT) mice; these effects were ameliorated in TLR2 KO mice. Likewise, ICH-induced blood-brain barrier (BBB) damage was also decreased in TLR2 KO mice. This effect was most likely due to reduced matrix metalloproteinase 9 (MMP9) activity in the TLR2 KO mice compared to WT mice. In primary astrocytes, hemin directly induced MMP9 activity as well as proinflammatory cytokine and chemokine expression in a TLR2-dependent manner. Finally, hemin-induced MMP9 activity and proinflammatory gene expression were almost completely blocked by TLR2-neutralizing antibodies. Taken together, our data propose that heme released to the brain parenchyma after ICH injury activates TLR2 in astrocytes and induces inflammatory gene expression and BBB damage, which contribute to secondary brain damage after ICH. [ABSTRACT FROM AUTHOR]

Background: The inflammatory myeloid cell activation is one of the hallmarks of experimental autoimmune encephalomyelitis (EAE), yet the in vivo role of the inflammatory myeloid cell activation in EAE has not been clearly resolved. It is well-known that IKK/NF-κB is a key signaling pathway that regulates inflammatory myeloid activation. Methods: We investigated the in vivo role of inflammatory myeloid cell activation in myelin oligodendrocyte glycoprotein (MOG) peptides-induced EAE using myeloid cell type-specific ikkβ gene conditional knockout-mice (LysM-Cre/IkkβF/F). Results: In our study, LysM-Cre/IkkβF/F mice had alleviated clinical signs of EAE corresponding to the decreased spinal demyelination, microglial activation, and immune cell infiltration in the spinal cord, compared to the wild-type mice (WT, IkkβF/F). Myeloid ikkβ gene deletion significantly reduced the percentage of CD4+/IFN-γ+ (Th1) and CD4+ /IL-17+ (Th17) cells but increased the percentages of CD4+ /CD25+/Foxp3+ (Treg) cells in the spinal cord and lymph nodes, corresponding to the altered mRNA expression of IFN-γ, IL-17, IL-23, and Foxp3 in the spinal cords of LysM-Cre/IkkβF/F EAE mice. Also, the beneficial effect of myeloid IKKβ deletion in EAE corresponded to the decreased permeability of the blood brain barrier (BBB). Conclusions: Our findings strongly suggest that IKK/NF-κB-induced myeloid cell activation exacerbates EAE by activating Th1 and Th17 responses and compromising the BBB. The development of NF-κB inhibitory agents with high efficacy through specific targeting of IKKβ in myeloid cells might be of therapeutic potential in MS and other autoimmune disorders. [ABSTRACT FROM AUTHOR]

Background: Intracerebral hemorrhage (ICH) is one of the major causes of stroke. After onset of ICH, massive infiltration of macrophages is detected in the peri-hematoma regions. Still, the function of these macrophages in ICH has not been completely elucidated. Results: In a collagenase-induced ICH model, CX3CR1+ macrophages accumulated in the peri-hematoma region. Characterization of these macrophages revealed expression of alternatively activated (M2) macrophage markers. In the macrophage-depleted mice, ICH-induced brain lesion volume was larger and neurological deficits were more severe compared to those of control mice, indicating a protective role of these macrophages in ICH. In the ICH-injured brain, mannose receptor-expressing macrophages increased at a delayed time point after ICH, indicating M2 polarization of the brain-infiltrating macrophages in the brain microenvironment. To explore this possibility, bone marrow-derived macrophages (BMDM) were co-cultured with mouse brain glial cells and then tested for activation phenotype. Upon co-culture with glia, the number of mannose receptor-positive M2 macrophages was significantly increased. Furthermore, treatment with glia-conditioned media increased the number of BMDM of M2 phenotype. Conclusions: In this study, our data suggest that brain-infiltrating macrophages after ICH are polarized to the M2 phenotype by brain glial cells and thereby contribute to recovery from ICH injury. [ABSTRACT FROM AUTHOR]

Background: The innate immune response plays an important role in the pathogenesis of intracerebral hemorrhage (ICH). Recent studies have shown that Toll-like receptor 2 (TLR2) is involved in the innate immune response in various neurological diseases, yet neither its role in ICH nor the mechanisms by which it functions have yet been elucidated. We examined these in this study using a collagenase-induced mouse ICH model with TLR2 knock-out (KO) mice. Results: TLR2 expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. Brain injury volume and neurological deficits following ICH were reduced in TLR2 KO mice compared to wild-type (WT) control mice. Heterologous blood-transfer experiments show that TLR2 signaling in brain-resident cells, but not leukocytes, contributes to the injury. In our study to elucidate underlying mechanisms, we found that damage to blood-brain-barrier (BBB) integrity following ICH was attenuated in TLR2 KO mice compared to WT mice, which may be due to reduced matrix metalloproteinase-9 (MMP9) activation in astrocytes. The reduced BBB damage accompanies decreased neutrophil infiltration and proinflammatory gene expression in the injured brain parenchyma, which may account for the attenuated brain damage in TLR2 KO mice after ICH. Conclusions: TLR2 plays a detrimental role in ICH-induced brain damage by activating MMP9 in astrocytes, compromising BBB, and enhancing neutrophils infiltration and proinflammatory gene expression. [ABSTRACT FROM AUTHOR]

Background Recent studies have indicated that Toll-like receptor 4 (TLR4), a pathogen-recognition receptor that triggers inflammatory signals in innate immune cells, is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. However, the possible function of sensory neuron TLR4 has not yet been formally addressed. In this regard, we investigated the role of TLR4 in itch signal transmission. Results TLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in the TLR4- deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated that TLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents. Conclusions Our data show that TLR4 on sensory neurons enhances histamine-induced itch signal transduction by potentiating TRPV1 activity. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation. [ABSTRACT FROM AUTHOR]

Background: Activated microglia elicits a robust amount of pro-inflammatory cytokines, which are implicated in the pathogenesis of tuberculosis in the central nervous system (CNS). However, little is known about the intracellular signaling mechanisms governing these inflammatory responses in microglia in response to Mycobacterium tuberculosis (Mtb). Methods: Murine microglial BV-2 cells and primary mixed glial cells were stimulated with sonicated Mtb (s-Mtb). Intracellular ROS levels were measured by staining with oxidative fluorescent dyes [2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE)]. NADPH oxidase activities were measured by lucigenin chemiluminescence assay. S-Mtb-induced MAPK activation and pro-inflammatory cytokine release in microglial cells were measured using by Western blot analysis and enzyme-linked immunosorbent assay, respectively. Results: We demonstrate that s-Mtb promotes the up-regulation of reactive oxygen species (ROS) and the rapid activation of mitogen-activated protein kinases (MAPKs), including p38 and extracellular signal-regulated kinase (ERK) ½, as well as the secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12p40 in murine microglial BV-2 cells and primary mixed glial cells. Both NADPH oxidase and mitochondrial electron transfer chain subunit I play an indispensable role in s-Mtb-induced MAPK activation and pro-inflammatory cytokine production in BV-2 cells and mixed glial cells. Furthermore, the activation of cytosolic NADPH oxidase p47phox and MAPKs (p38 and ERK1/2) is mutually dependent on s-Mtb-induced inflammatory signaling in murine microglia. Neither TLR2 nor dectin-1 was involved in s-Mtb-induced inflammatory responses in murine microglia. Conclusion: These data collectively demonstrate that s-Mtb actively induces the pro-inflammatory response in microglia through NADPH oxidase-dependent ROS generation, although the specific pattern-recognition receptors involved in these responses remain to be identified. [ABSTRACT FROM AUTHOR]