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Plexin-A1 is required for Toll-like receptor-mediated microglial activation in the development of lipopolysaccharide-induced encephalopathy.

Ito T, Yoshida K, Negishi T, Miyajima M, Takamatsu H, Kikutani H, Kumanogoh A, Yukawa K - Int. J. Mol. Med. (2014)

Bottom Line: In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control.These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia.Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan.

ABSTRACT
Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (-/-) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1β and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1-/- mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1β or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1-/- mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

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Expression of Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in hippocampus of WT and Plexin-A1−/− mice administered lipopolysaccharide. WT or Plexin-A1−/− mice were subjected to lateral ventricular injection with saline or lipopolysaccharide (LPS; 200 μg/kg). Western blotting detected Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in the mouse hippocampus in all experimental conditions except for Plexin-A1 in the Plexin-A1−/− hippocampus. Nrp1, Neuropilin-1; TLR4, Toll-like receptor 4; VEH, vehicle (saline); LPS, lipopolysaccharide; WT, wild-type; A1−/−, Plexin-A1−/−.
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f4-ijmm-33-05-1122: Expression of Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in hippocampus of WT and Plexin-A1−/− mice administered lipopolysaccharide. WT or Plexin-A1−/− mice were subjected to lateral ventricular injection with saline or lipopolysaccharide (LPS; 200 μg/kg). Western blotting detected Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in the mouse hippocampus in all experimental conditions except for Plexin-A1 in the Plexin-A1−/− hippocampus. Nrp1, Neuropilin-1; TLR4, Toll-like receptor 4; VEH, vehicle (saline); LPS, lipopolysaccharide; WT, wild-type; A1−/−, Plexin-A1−/−.

Mentions: LPS activates microglia through its binding to the LPS receptor, TLR4, on the microglial cell surface (5,6). Plexin-A1−/− microglia may be overactivated in the LPS response due to their inability to use Sema3A to induce apoptosis of activated microglia (28). To test this hypothesis, we compared the microglial responses of WT and Plexin-A1−/− mice following acute administration of LPS to the lateral ventricles. Direct LPS ICV injection led to robust neuroinflammatory responses in the brain, not through activation of peripheral inflammatory cells such as macrophages, but through direct microglial activation (30,31). Immunoblotting studies confirmed the expression of Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in the hippocampus 18 h after ICV injection of saline or LPS (Fig. 4). To elucidate whether the deletion of Plexin-A1 would affect classical microglial activation and the associated inflammatory response, we examined the number of microglia and the expression level of inflammation-related mediators. Of note, 18 h after the ICV injection of LPS, the number of Iba-1-positive cells did not increase significantly in Plexin-A1−/− mice compared with WT mice (Fig. 5), and there was no significant increase in inflammation-related mediators such as COX-2, IL-1β, TNF-α and iNOS (Fig. 6). Thus, levels of brain inflammation after ICV injection of LPS were significantly reduced in Plexin-A1−/− mice as compared to WT mice. During neuroinflammation, changes in vascular permeability lead to the development of brain edema, which in turn results in the enlargement of the lateral ventricles of the brain (32). H&E-stained coronal brain sections revealed that the ratio of the lateral ventricular area to the cerebral hemisphere ipsilateral to the injection site was significantly larger in LPS-injected WT mice as compared to saline-treated mice (Fig. 7A). However, significant enlargement of the lateral ventricle following LPS administration was not observed in Plexin-A1−/− mice (Fig. 7B). Neutrophil leukocytes migrate into the CNS towards various inflammatory stimuli that exacerbate brain tissue damage through the release of inflammation-related mediators and through an increase in vascular permeability (33,34). To examine whether the absence of Plexin-A1 affected neutrophil recruitment, we quantified their infiltration using an esterase stain 18 h after ICV administration of LPS to specifically mark neutrophils. Following administration, the number of esterase-positive cells was significantly fewer in the Plexin-A1−/− cortex as compared to the WT controls (Fig. 7B). Mice administered saline did not show any significant difference based on genotype (Fig. 7B).


Plexin-A1 is required for Toll-like receptor-mediated microglial activation in the development of lipopolysaccharide-induced encephalopathy.

Ito T, Yoshida K, Negishi T, Miyajima M, Takamatsu H, Kikutani H, Kumanogoh A, Yukawa K - Int. J. Mol. Med. (2014)

Expression of Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in hippocampus of WT and Plexin-A1−/− mice administered lipopolysaccharide. WT or Plexin-A1−/− mice were subjected to lateral ventricular injection with saline or lipopolysaccharide (LPS; 200 μg/kg). Western blotting detected Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in the mouse hippocampus in all experimental conditions except for Plexin-A1 in the Plexin-A1−/− hippocampus. Nrp1, Neuropilin-1; TLR4, Toll-like receptor 4; VEH, vehicle (saline); LPS, lipopolysaccharide; WT, wild-type; A1−/−, Plexin-A1−/−.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4020473&req=5

f4-ijmm-33-05-1122: Expression of Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in hippocampus of WT and Plexin-A1−/− mice administered lipopolysaccharide. WT or Plexin-A1−/− mice were subjected to lateral ventricular injection with saline or lipopolysaccharide (LPS; 200 μg/kg). Western blotting detected Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in the mouse hippocampus in all experimental conditions except for Plexin-A1 in the Plexin-A1−/− hippocampus. Nrp1, Neuropilin-1; TLR4, Toll-like receptor 4; VEH, vehicle (saline); LPS, lipopolysaccharide; WT, wild-type; A1−/−, Plexin-A1−/−.
Mentions: LPS activates microglia through its binding to the LPS receptor, TLR4, on the microglial cell surface (5,6). Plexin-A1−/− microglia may be overactivated in the LPS response due to their inability to use Sema3A to induce apoptosis of activated microglia (28). To test this hypothesis, we compared the microglial responses of WT and Plexin-A1−/− mice following acute administration of LPS to the lateral ventricles. Direct LPS ICV injection led to robust neuroinflammatory responses in the brain, not through activation of peripheral inflammatory cells such as macrophages, but through direct microglial activation (30,31). Immunoblotting studies confirmed the expression of Plexin-A1, Neuropilin-1, Sema3A, and TLR4 in the hippocampus 18 h after ICV injection of saline or LPS (Fig. 4). To elucidate whether the deletion of Plexin-A1 would affect classical microglial activation and the associated inflammatory response, we examined the number of microglia and the expression level of inflammation-related mediators. Of note, 18 h after the ICV injection of LPS, the number of Iba-1-positive cells did not increase significantly in Plexin-A1−/− mice compared with WT mice (Fig. 5), and there was no significant increase in inflammation-related mediators such as COX-2, IL-1β, TNF-α and iNOS (Fig. 6). Thus, levels of brain inflammation after ICV injection of LPS were significantly reduced in Plexin-A1−/− mice as compared to WT mice. During neuroinflammation, changes in vascular permeability lead to the development of brain edema, which in turn results in the enlargement of the lateral ventricles of the brain (32). H&E-stained coronal brain sections revealed that the ratio of the lateral ventricular area to the cerebral hemisphere ipsilateral to the injection site was significantly larger in LPS-injected WT mice as compared to saline-treated mice (Fig. 7A). However, significant enlargement of the lateral ventricle following LPS administration was not observed in Plexin-A1−/− mice (Fig. 7B). Neutrophil leukocytes migrate into the CNS towards various inflammatory stimuli that exacerbate brain tissue damage through the release of inflammation-related mediators and through an increase in vascular permeability (33,34). To examine whether the absence of Plexin-A1 affected neutrophil recruitment, we quantified their infiltration using an esterase stain 18 h after ICV administration of LPS to specifically mark neutrophils. Following administration, the number of esterase-positive cells was significantly fewer in the Plexin-A1−/− cortex as compared to the WT controls (Fig. 7B). Mice administered saline did not show any significant difference based on genotype (Fig. 7B).

Bottom Line: In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control.These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia.Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan.

ABSTRACT
Recent investigations have suggested that semaphorins, which are known repulsive axon guidance molecules, may play a crucial role in maintaining brain homeostasis by regulating microglial activity. Sema3A, secreted in higher amounts from injured neurons, is considered to suppress excessive inflammatory responses by inducing microglial apoptosis through its binding to Plexin-A1 receptors on activated microglia. To clarify the in vivo role of Plexin-A1-mediated signaling in lipopolysaccharide (LPS)-induced injury in mouse brain, we examined the neuroinflammatory changes initiated by LPS administration to the cerebral ventricles of wild-type (WT) and Plexin-A1-deficient (-/-) mice. WT mice administered LPS exhibited a significantly higher expression of COX-2, iNOS, IL-1β and TNF-α in the hippocampus, and a significantly greater ventricular enlargement and intracerebral infiltration of leukocytes, as compared with the saline-treated group. By contrast, Plexin-A1-/- mice administered LPS did not exhibit a significantly increased expression of COX-2, iNOS, IL-1β or TNF-α in the hippocampus as compared with the saline-treated group. Plexin-A1-/- mice administered LPS did not show significant increases in ventricle size or infiltration of leukocytes into the brain, as compared with the saline-treated group. In WT, but not in the Plexin-A1-/- primary microglia treated with LPS, Sema3A induced significantly more nitric oxide production than in the immunoglobulin G control. These results revealed the crucial role of the Sema3A-Plexin-A1 interaction in the Toll-like receptor 4-mediated signaling of the LPS-induced activation of microglia. Thus, results of the present study revealed the essential role of Plexin-A1 in the development of LPS-induced neuroinflammation in mice, suggesting the possible application of microglial control of the semaphorin-plexin signaling system to the treatment of LPS-induced encephalopathy and other psychiatric diseases associated with neuroinflammation.

Show MeSH
Related in: MedlinePlus