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The induction of neuronal death by up-regulated microglial cathepsin H in LPS-induced neuroinflammation.

Fan K, Li D, Zhang Y, Han C, Liang J, Hou C, Xiao H, Ikenaka K, Ma J - J Neuroinflammation (2015)

Bottom Line: Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level.Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation.The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. fankaijunjun@hotmail.com.

ABSTRACT

Background: Neuroinflammation is a hallmark that leads to selective neuronal loss and/or dysfunction in neurodegenerative disorders. Microglia-derived lysosomal cathepsins are increasingly recognized as important inflammatory mediators to trigger signaling pathways that aggravate neuroinflammation. However, cathepsin H (Cat H), a cysteine protease, has been far less studied in neuroinflammation, compared to cathepsins B, D, L, and S. The expression patterns and functional roles of Cat H in the brain in neuroinflammation remain unknown.

Methods: C57BL/6J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg). Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to analyze expression and localization of Cat H in the brain. Nitrite assay was used to examine microglial activation in vitro; ELISA was used to determine the release of Cat H and proinflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ). Cat H activity was analyzed by cellular Cat H assay kit. Flow cytometry and in situ cell death detection were used to investigate neuronal death. Data were evaluated for statistical significance with one-way ANOVA and t test.

Results: Cat H mRNA was only present in perivascular microglia and non-parenchymal sites under normal conditions. After LPS injection, Cat H mRNA expression in activated microglia in different brain regions was increased. Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level. The expression of Cat H protein exhibited the similar alterations after LPS injection. In vitro, inflammatory stimulation (LPS, TNF-α, IL-1β, IL-6, and IFN-γ) increased the release and activity of Cat H in microglia. Conversely, addition of Cat H to microglia promoted the production and release of NO, IL-1β, and IFN-γ which could be prevented by neutralizing antibody. Further, addition of Cat H to Neuro2a cells induced neuronal death.

Conclusions: Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation. The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.

No MeSH data available.


Related in: MedlinePlus

Proinflammatory cytokines induced microglial Cat H release and up-regulated Cat H activityin vitro. The level of Cat H in the media of BV2 cells and primary microglia after various inflammatory treatments for 24 h was measured by ELISA and shown in (A-E). The activity of Cat H in the media of BV-2 cells and primary microglia after various inflammatory treatments was measured and shown in (F). Data were expressed as mean + SEM from three independent experiments. *, #: P < 0.05; **: P < 0.01, NS: no significance in (A-E), *, #: P < 0.05 vs control in (F).
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Fig3: Proinflammatory cytokines induced microglial Cat H release and up-regulated Cat H activityin vitro. The level of Cat H in the media of BV2 cells and primary microglia after various inflammatory treatments for 24 h was measured by ELISA and shown in (A-E). The activity of Cat H in the media of BV-2 cells and primary microglia after various inflammatory treatments was measured and shown in (F). Data were expressed as mean + SEM from three independent experiments. *, #: P < 0.05; **: P < 0.01, NS: no significance in (A-E), *, #: P < 0.05 vs control in (F).

Mentions: Then, we examined the Cat H release in the culture media of BV2 cells or primary microglia by ELISA. We observed the low level of Cat H content in the media in absence of proinflammatory stimuli, suggesting that there is a basal level of Cat H production and secretion in untreated microglia. In contrast, the levels of Cat H were significantly increased in the media of BV2 cells following TNF-α, IL-1β, IL-6, and IFN-γ treatments for 24 h, respectively. Among these, TNF-α and IFN-γ induced Cat H increase in a dose-dependent manner (Figure 3B,E). Besides, IL-6 treatment, to various degrees, induced Cat H release in primary microglia (Figure 3D). However, LPS has little effect on Cat H release in either BV2 or primary microglia (Figure 3A). These results suggest an inducible effect of proinflammatory cytokines on Cat H release in microglia.Figure 3


The induction of neuronal death by up-regulated microglial cathepsin H in LPS-induced neuroinflammation.

Fan K, Li D, Zhang Y, Han C, Liang J, Hou C, Xiao H, Ikenaka K, Ma J - J Neuroinflammation (2015)

Proinflammatory cytokines induced microglial Cat H release and up-regulated Cat H activityin vitro. The level of Cat H in the media of BV2 cells and primary microglia after various inflammatory treatments for 24 h was measured by ELISA and shown in (A-E). The activity of Cat H in the media of BV-2 cells and primary microglia after various inflammatory treatments was measured and shown in (F). Data were expressed as mean + SEM from three independent experiments. *, #: P < 0.05; **: P < 0.01, NS: no significance in (A-E), *, #: P < 0.05 vs control in (F).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4379721&req=5

Fig3: Proinflammatory cytokines induced microglial Cat H release and up-regulated Cat H activityin vitro. The level of Cat H in the media of BV2 cells and primary microglia after various inflammatory treatments for 24 h was measured by ELISA and shown in (A-E). The activity of Cat H in the media of BV-2 cells and primary microglia after various inflammatory treatments was measured and shown in (F). Data were expressed as mean + SEM from three independent experiments. *, #: P < 0.05; **: P < 0.01, NS: no significance in (A-E), *, #: P < 0.05 vs control in (F).
Mentions: Then, we examined the Cat H release in the culture media of BV2 cells or primary microglia by ELISA. We observed the low level of Cat H content in the media in absence of proinflammatory stimuli, suggesting that there is a basal level of Cat H production and secretion in untreated microglia. In contrast, the levels of Cat H were significantly increased in the media of BV2 cells following TNF-α, IL-1β, IL-6, and IFN-γ treatments for 24 h, respectively. Among these, TNF-α and IFN-γ induced Cat H increase in a dose-dependent manner (Figure 3B,E). Besides, IL-6 treatment, to various degrees, induced Cat H release in primary microglia (Figure 3D). However, LPS has little effect on Cat H release in either BV2 or primary microglia (Figure 3A). These results suggest an inducible effect of proinflammatory cytokines on Cat H release in microglia.Figure 3

Bottom Line: Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level.Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation.The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Dalian Medical University, West Section No. 9, South Road, Lvshun, Dalian, 116044, , Liaoning, China. fankaijunjun@hotmail.com.

ABSTRACT

Background: Neuroinflammation is a hallmark that leads to selective neuronal loss and/or dysfunction in neurodegenerative disorders. Microglia-derived lysosomal cathepsins are increasingly recognized as important inflammatory mediators to trigger signaling pathways that aggravate neuroinflammation. However, cathepsin H (Cat H), a cysteine protease, has been far less studied in neuroinflammation, compared to cathepsins B, D, L, and S. The expression patterns and functional roles of Cat H in the brain in neuroinflammation remain unknown.

Methods: C57BL/6J mice were intraperitoneally injected with either 0.9% saline or lipopolysaccharide (LPS, 5 mg/kg). Immunohistochemistry (IHC) and in situ hybridization (ISH) were used to analyze expression and localization of Cat H in the brain. Nitrite assay was used to examine microglial activation in vitro; ELISA was used to determine the release of Cat H and proinflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ). Cat H activity was analyzed by cellular Cat H assay kit. Flow cytometry and in situ cell death detection were used to investigate neuronal death. Data were evaluated for statistical significance with one-way ANOVA and t test.

Results: Cat H mRNA was only present in perivascular microglia and non-parenchymal sites under normal conditions. After LPS injection, Cat H mRNA expression in activated microglia in different brain regions was increased. Twenty-four hours after LPS injection, Cat H mRNA expression was maximal in SNr; 72 h later, it peaked in cerebral cortex and hippocampus then decreased and maintained at a low level. The expression of Cat H protein exhibited the similar alterations after LPS injection. In vitro, inflammatory stimulation (LPS, TNF-α, IL-1β, IL-6, and IFN-γ) increased the release and activity of Cat H in microglia. Conversely, addition of Cat H to microglia promoted the production and release of NO, IL-1β, and IFN-γ which could be prevented by neutralizing antibody. Further, addition of Cat H to Neuro2a cells induced neuronal death.

Conclusions: Taken together, these data indicate that the up-regulated microglial Cat H expression, release, and activity in the brain lead to neuronal death in neuroinflammation. The functional link of Cat H with microglial activation might contribute to the initiation and maintenance of microglia-driven chronic neuroinflammation.

No MeSH data available.


Related in: MedlinePlus