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The role of toll-like receptor 9 in chronic stress-induced apoptosis in macrophage.

Xiang Y, Yan H, Zhou J, Zhang Q, Hanley G, Caudle Y, LeSage G, Zhang X, Yin D - PLoS ONE (2015)

Bottom Line: Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival.We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress.TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, United States of America; Department of Pharmacology, Shandong University School of Medicine, Jinan, People's Republic of China.

ABSTRACT
Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.

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TLR9 deficiency attenuates chronic stress-induced changes of apoptosis related pathways TLR9 knockout mice or wild type BALB/c mice aged 6 to 8 weeks were subjected to a 12 h physical restraint daily.After 2 d stress, mice were sacrificed by cervical dislocation, and the macrophages were harvested, purified and cultured (5 × 105 cells/well) on culture plates for 24 hours. The expression of total and phospho-p38, total and phospho-Akt, total and phospho-GSK-3β were analyzed by Western blot. Means and SEs were calculated from 7 mice per group. *p < 0.05, **p< 0.01 compared with indicated groups.
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pone.0123447.g006: TLR9 deficiency attenuates chronic stress-induced changes of apoptosis related pathways TLR9 knockout mice or wild type BALB/c mice aged 6 to 8 weeks were subjected to a 12 h physical restraint daily.After 2 d stress, mice were sacrificed by cervical dislocation, and the macrophages were harvested, purified and cultured (5 × 105 cells/well) on culture plates for 24 hours. The expression of total and phospho-p38, total and phospho-Akt, total and phospho-GSK-3β were analyzed by Western blot. Means and SEs were calculated from 7 mice per group. *p < 0.05, **p< 0.01 compared with indicated groups.

Mentions: Accumulating evidence indicates that p38 MAPK participates as a modulator in Bcl-2/Bax-mediated apoptosis in neuroblastoma cells [28] [29]. It also has been demonstrated that activated Akt alters the ratio of Bcl-2 and Bax and exhibits an anti-apoptotic role in various cells [30]. Additionally, recent studies have revealed cross-talk between TLR signaling and the Akt/GSK-3β or p38 MAPK signaling pathway [13] [31]. To examine whether chronic stress activates p38 MAPK and Akt/GSK-3β signaling in TLR9-mediated signaling, the levels of phosphorylated p38 (phospho-p38), phospho-Akt and phospho-GSK-3β in macrophages following stress treatment were examined by western blot analysis. The results of the present study confirmed that chronic stress promoted p38 phosphorylation in wild type macrophage. Moreover, stress-induced p38 MAPK activation was reversed in TLR9 knockout macrophages suggesting that stress markedly increases the level of phospho-p38 through TLR9 (Fig 6). We also found that the increasing levels of phospho-Akt and phospho-GSK-3β were significantly abolished by chronic stress in wild type macrophages but not in TLR9 deficient macrophages, demonstrating that chronic stress decreases the activation of phospho-Akt/phospho-GSK-3β signaling in a TLR9-dependent manner (Fig 6).


The role of toll-like receptor 9 in chronic stress-induced apoptosis in macrophage.

Xiang Y, Yan H, Zhou J, Zhang Q, Hanley G, Caudle Y, LeSage G, Zhang X, Yin D - PLoS ONE (2015)

TLR9 deficiency attenuates chronic stress-induced changes of apoptosis related pathways TLR9 knockout mice or wild type BALB/c mice aged 6 to 8 weeks were subjected to a 12 h physical restraint daily.After 2 d stress, mice were sacrificed by cervical dislocation, and the macrophages were harvested, purified and cultured (5 × 105 cells/well) on culture plates for 24 hours. The expression of total and phospho-p38, total and phospho-Akt, total and phospho-GSK-3β were analyzed by Western blot. Means and SEs were calculated from 7 mice per group. *p < 0.05, **p< 0.01 compared with indicated groups.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401452&req=5

pone.0123447.g006: TLR9 deficiency attenuates chronic stress-induced changes of apoptosis related pathways TLR9 knockout mice or wild type BALB/c mice aged 6 to 8 weeks were subjected to a 12 h physical restraint daily.After 2 d stress, mice were sacrificed by cervical dislocation, and the macrophages were harvested, purified and cultured (5 × 105 cells/well) on culture plates for 24 hours. The expression of total and phospho-p38, total and phospho-Akt, total and phospho-GSK-3β were analyzed by Western blot. Means and SEs were calculated from 7 mice per group. *p < 0.05, **p< 0.01 compared with indicated groups.
Mentions: Accumulating evidence indicates that p38 MAPK participates as a modulator in Bcl-2/Bax-mediated apoptosis in neuroblastoma cells [28] [29]. It also has been demonstrated that activated Akt alters the ratio of Bcl-2 and Bax and exhibits an anti-apoptotic role in various cells [30]. Additionally, recent studies have revealed cross-talk between TLR signaling and the Akt/GSK-3β or p38 MAPK signaling pathway [13] [31]. To examine whether chronic stress activates p38 MAPK and Akt/GSK-3β signaling in TLR9-mediated signaling, the levels of phosphorylated p38 (phospho-p38), phospho-Akt and phospho-GSK-3β in macrophages following stress treatment were examined by western blot analysis. The results of the present study confirmed that chronic stress promoted p38 phosphorylation in wild type macrophage. Moreover, stress-induced p38 MAPK activation was reversed in TLR9 knockout macrophages suggesting that stress markedly increases the level of phospho-p38 through TLR9 (Fig 6). We also found that the increasing levels of phospho-Akt and phospho-GSK-3β were significantly abolished by chronic stress in wild type macrophages but not in TLR9 deficient macrophages, demonstrating that chronic stress decreases the activation of phospho-Akt/phospho-GSK-3β signaling in a TLR9-dependent manner (Fig 6).

Bottom Line: Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival.We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress.TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, United States of America; Department of Pharmacology, Shandong University School of Medicine, Jinan, People's Republic of China.

ABSTRACT
Emerging evidence implied that chronic stress has been exerting detrimental impact on immune system functions in both humans and animals. Toll-like receptors (TLRs) have been shown to play an essential role in modulating immune responses and cell survival. We have recently shown that TLR9 deficiency protects against lymphocyte apoptosis induced by chronic stress. However, the exact role of TLR9 in stress-mediated change of macrophage function remains unclear. The results of the current study showed that when BALB/c mice were treated with restraint stress (12 h daily for 2 days), the number of macrophages recruited to the peritoneal cavity was obviously increased. Results also demonstrated that the sustained effects of stress elevated cytokine IL-1β, TNF-α and IL-10 production yet diminished IFN-γ production from macrophage, which led to apoptotic cell death. However, TLR9 deficiency prevented the chronic stress-mediated accumulation of macrophages. In addition, knocking out TLR9 significantly abolished the chronic stress-induced imbalance of cytokine levels and apoptosis in macrophage. TLR9 deficiency was also found to reverse elevation of plasma IL-1β, IL-10 and IL-17 levels and decrease of plasma IFN-γ level under the condition of chronic stress. These results indicated that TLR9-mediated macrophage responses were required for chronic stress-induced immunosuppression. Further exploration showed that TLR9 deficiency prevented the increment of p38 MAPK phosphorylation and reduction of Akt/Gsk-3β phosphorylation; TLR9 deficiency also attenuated the release of mitochondrial cytochrome c into cytoplasm, caused upregulation of Bcl-2/Bax protein ratio, downregulation of cleavage of caspase-3 and PARP, as well as decreased TUNEL-positive cells in macrophage of stressed mice. Collectively, our studies demonstrated that deficiency of TLR9 maintained macrophage function by modulating macrophage accumulation and attenuating macrophage apoptosis, thus preventing immunosuppression in restraint-stressed mice.

Show MeSH
Related in: MedlinePlus