Neuronal toll-like receptor 4 signaling induces brain endothelial activation and neutrophil transmigration in vitro

Abstract

BackgroundThe innate immune response in the brain is initiated by pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) produced in response to central nervous system (CNS) infection or injury. These molecules activate members of the Toll-like receptor (TLR) family, of which TLR4 is the receptor for bacterial lipopolysaccharide (LPS). Although neurons have been reported to express TLR4, the function of TLR4 activation in neurons remains unknown.MethodsTLR4 mRNA expression in primary mouse glial and neuronal cultures was assessed by RT-PCR. Mouse mixed glial, neuronal or endothelial cell cultures were treated with LPS in the absence or the presence of a TLR4 specific antagonist (VIPER) or a specific JNK inhibitor (SP600125). Expression of inflammatory mediators was assayed by cytometric bead array (CBA) and ELISA. Activation of extracellular-signal regulated kinase 1/2 (ERK1/2), p38, c-Jun-N-terminal kinase (JNK) and c-Jun was assessed by Western blot. The effect of conditioned media of untreated- versus LPS-treated glial or neuronal cultures on endothelial activation was assessed by neutrophil transmigration assay, and immunocytochemistry and ELISA were used to measure expression of intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1).ResultsLPS induces strong release of the chemokines RANTES and CXCL1 (KC), tumor necrosis factor-α (TNFα) and IL-6 in primary mouse neuronal cultures. In contrast, LPS induced release of IL-1α, IL-1β and granulocyte-colony stimulating factor (G-CSF) in mixed glial, but not in neuronal cultures. LPS-induced neuronal KC expression and release were completely blocked by VIPER. In glial cultures, LPS induced activation of ERK1/2, p38 and JNK. In contrast, in neuronal cultures, LPS activated JNK but not ERK1/2 or p38, and the specific JNK inhibitor SP600125 significantly blocked LPS-induced KC expression and release. Finally, conditioned medium of LPS-treated neuronal cultures induced strong expression of ICAM-1 and VCAM-1 on endothelial cells, and induced infiltration of neutrophils across the endothelial monolayer, which was inhibited by VIPER.ConclusionThese data demonstrate for the first time that neurons can play a role as key sensors of infection to initiate CNS inflammation.

Cross talk between AT1receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus

“…These results are in accordance with previous studies showing that ANG II promotes the involvement of microglia in NADPH-derived ROS in primary mesencephalic cultures (63) and that lipopolysaccharide failed to induce ROS production in bone marrow-derived macrophages cells from the same TLR4-deficient mice (67). Nonetheless, TLR4 in the brain is not exclusively expressed in microglia, and, along with AT 1a Rs, TLR4 has been shown to be also present in neurons (40,48,64). Similarly, the NADPH oxidase family, a primary source contributing to ANG II-mediated superoxide production, is present in both microglia as well as neurons (12,55,70,75,85).…”

“…Taken together, our results support a major contribution of microglia and TLR4 to ANG II-mediated ROS production within the PVN. However, in addition to their presence in microglia [as shown in the present study and elsewhere (38,68)], TLR4 as well as AT 1a Rs have also been shown to be expressed in neurons (40,48,64). Thus, to further test the relative contribution of microglia versus neurons as cell targets mediating ANG II effects on PVN ROS production, we performed similar experiments in mice in which AT 1a Rs were specifically deleted from PVN neurons [(Sim1)-AT 1a KO mice; see METHODS].…”

Lipocalin 2 modulates the cellular response to amyloid beta

“…15,17 Of relevance, the expression of most of these proinflammatory molecules, including LCN2, can be upregulated through Toll-like receptor 4, 42 which is activated by LPS, but is also closely involved in the binding of extracellular Ab peptides. 43 Toll-like receptor 4 signaling cascades, which are distinct between glial cells and neurons, 44 might also explain the differences that we observe concerning the production of LCN2, a topic that warrants further investigation. Accordingly, it was recently shown that astrocytes isolated from the brain of Lcn2( À / À ) mice are unable to express the classical proinflammatory genes in response to LPS and interferon-g. 32 We observed the same kind of gene expression abrogation in Lcn2( À / À ) astrocytes after treatment with Ab , which may denote an anti-inflammatory phenotype that is conferring protection in response to these toxic peptides (Figure 7).…”

Heme Oxygenase-1 Promotes Neuron Survival through Down-Regulation of Neuronal NLRP1 Expression after Spinal Cord Injury

“…Another possibility is that TLRs might induce post-SCI NLRP1 inflammasome formation. Although TLRs are generally considered to be expressed in microglia and astrocytes, recent research has showed that TLR expression in neurons is pivotal under both physiological and pathological conditions [54][55][56][57]. Perhaps SCI changes expression of certain TLRs which recognize danger signals after injury and subsequently causes inflammasome formation.…”