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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 deficency inhibits stress-induced change in caspase-3 and PARP activation and ratio of Bcl-2/Bax 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 cleaved caspase-3 and cleaved PARP (A), and Bcl-2/Bax (B) was 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.g005: TLR9 deficency inhibits stress-induced change in caspase-3 and PARP activation and ratio of Bcl-2/Bax 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 cleaved caspase-3 and cleaved PARP (A), and Bcl-2/Bax (B) was 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: The levels of major apoptosis-related proteins were detected to further assess the mechanisms underlying cellular changes observed in mice after stress challenge. We found that the levels of cleaved caspase-3 and cleaved PARP, two well-known characteristics of apoptosis, were remarkably increased in macrophages of wild type mice following stress treatment, whereas the increases of cleaved caspase-3 and cleaved PARP were attenuated markedly in TLR9 deficient macrophages (Fig 5A). As protein expressions of Bcl-2 and Bax are involved in the chronic stress-induced apoptotic pathway [27], we examined the ratio of Bcl-2 and Bax in macrophages to elucidate the mechanism of stress-induced apoptosis. Stress challenge markedly decreased the ratio of Bcl-2/Bax in wild type macrophages; moreover, the expressions of Bcl-2 and Bax in the macrophages were not altered in TLR9 deficient mice. Our data suggested that Bcl-2 family participate in TLR9-mediated macrophage signaling after stress treatment (Fig 5B).


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 deficency inhibits stress-induced change in caspase-3 and PARP activation and ratio of Bcl-2/Bax 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 cleaved caspase-3 and cleaved PARP (A), and Bcl-2/Bax (B) was 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.g005: TLR9 deficency inhibits stress-induced change in caspase-3 and PARP activation and ratio of Bcl-2/Bax 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 cleaved caspase-3 and cleaved PARP (A), and Bcl-2/Bax (B) was 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: The levels of major apoptosis-related proteins were detected to further assess the mechanisms underlying cellular changes observed in mice after stress challenge. We found that the levels of cleaved caspase-3 and cleaved PARP, two well-known characteristics of apoptosis, were remarkably increased in macrophages of wild type mice following stress treatment, whereas the increases of cleaved caspase-3 and cleaved PARP were attenuated markedly in TLR9 deficient macrophages (Fig 5A). As protein expressions of Bcl-2 and Bax are involved in the chronic stress-induced apoptotic pathway [27], we examined the ratio of Bcl-2 and Bax in macrophages to elucidate the mechanism of stress-induced apoptosis. Stress challenge markedly decreased the ratio of Bcl-2/Bax in wild type macrophages; moreover, the expressions of Bcl-2 and Bax in the macrophages were not altered in TLR9 deficient mice. Our data suggested that Bcl-2 family participate in TLR9-mediated macrophage signaling after stress treatment (Fig 5B).

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