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Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression.

Zha J, Smith A, Andreansky S, Bracchi-Ricard V, Bethea JR - J Neuroinflammation (2014)

Bottom Line: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production.The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells.Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Miami Project to Cure Paralysis, Department of Neurosurgery, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. VBracchi@med.miami.edu.

ABSTRACT

Background: Chronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.

Methods: To assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.

Results: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.

Conclusions: These studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.

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Exposure to norepinephrine (NE) in vitro impairs T-cell cytokine production in response to PMA/ionomycin stimulation. Enriched splenic T-cells (106 cells/ml) were cultured with 10 μM NE or its vehicle (Vehicle) in vitro. After two days of NE exposure, cells were stimulated with PMA/ionomycin for four hours in the presence of brefeldin A. Intracellular cytokine staining and flow cytometry analysis were performed to measure cytokine production. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (B) Bar graph represents the mean ± SEM percentages cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on lymphocytes were collected. Experiments were performed in triplicate, *P < 0.05, **P < 0.01, one-tailed Student’s t-test.
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Figure 10: Exposure to norepinephrine (NE) in vitro impairs T-cell cytokine production in response to PMA/ionomycin stimulation. Enriched splenic T-cells (106 cells/ml) were cultured with 10 μM NE or its vehicle (Vehicle) in vitro. After two days of NE exposure, cells were stimulated with PMA/ionomycin for four hours in the presence of brefeldin A. Intracellular cytokine staining and flow cytometry analysis were performed to measure cytokine production. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (B) Bar graph represents the mean ± SEM percentages cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on lymphocytes were collected. Experiments were performed in triplicate, *P < 0.05, **P < 0.01, one-tailed Student’s t-test.

Mentions: We next determined whether NE stimulation resulted in defective cytokine production in the enriched T-cells. Enriched T-cells were incubated for two days with NE prior to adding PMA/ionomycin and intracellular cytokine profile of these cells was characterized after 4 hours of stimulation. Compared with vehicle control, NE stimulation significantly reduced both IFN-γ (Vehicle: 4.9 ± 0.3%; NE: 3.5 ± 0.4%; P = 0.02) and TNF-α production (Vehicle: 3.5 ± 0.3%; NE: 1.1 ± 0.1%; P = 0.001) of CD4+ T-cells (Figure 10A, B), as well TNF-α production (Vehicle: 6.4 ± 0.4%; NE: 3.7 ± 0.7%; P = 0.01) in CD8+ T-cells (Figure 10C, D), These data strongly suggest that sustained elevated levels of splenic NE following chronic SCI could induce T-cell exhaustion and dysfunction of cytokine production.


Chronic thoracic spinal cord injury impairs CD8+ T-cell function by up-regulating programmed cell death-1 expression.

Zha J, Smith A, Andreansky S, Bracchi-Ricard V, Bethea JR - J Neuroinflammation (2014)

Exposure to norepinephrine (NE) in vitro impairs T-cell cytokine production in response to PMA/ionomycin stimulation. Enriched splenic T-cells (106 cells/ml) were cultured with 10 μM NE or its vehicle (Vehicle) in vitro. After two days of NE exposure, cells were stimulated with PMA/ionomycin for four hours in the presence of brefeldin A. Intracellular cytokine staining and flow cytometry analysis were performed to measure cytokine production. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (B) Bar graph represents the mean ± SEM percentages cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on lymphocytes were collected. Experiments were performed in triplicate, *P < 0.05, **P < 0.01, one-tailed Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4230802&req=5

Figure 10: Exposure to norepinephrine (NE) in vitro impairs T-cell cytokine production in response to PMA/ionomycin stimulation. Enriched splenic T-cells (106 cells/ml) were cultured with 10 μM NE or its vehicle (Vehicle) in vitro. After two days of NE exposure, cells were stimulated with PMA/ionomycin for four hours in the presence of brefeldin A. Intracellular cytokine staining and flow cytometry analysis were performed to measure cytokine production. (A) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD4+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (B) Bar graph represents the mean ± SEM percentages cytokine producing CD4+ T-cell in response to PMA/ionomycin stimulation. (C) Representative dot plots show the percentage of IFN-γ+ cells and TNF-α+ cells in gated CD8+ T-cells following PMA/ionomycin stimulation or with brefeldin A only (unstimulated). (D) Bar graph represents the mean ± SEM percentages of cytokine producing CD8+ T-cells in response to PMA/ionomycin stimulation. Ten thousand events gated on lymphocytes were collected. Experiments were performed in triplicate, *P < 0.05, **P < 0.01, one-tailed Student’s t-test.
Mentions: We next determined whether NE stimulation resulted in defective cytokine production in the enriched T-cells. Enriched T-cells were incubated for two days with NE prior to adding PMA/ionomycin and intracellular cytokine profile of these cells was characterized after 4 hours of stimulation. Compared with vehicle control, NE stimulation significantly reduced both IFN-γ (Vehicle: 4.9 ± 0.3%; NE: 3.5 ± 0.4%; P = 0.02) and TNF-α production (Vehicle: 3.5 ± 0.3%; NE: 1.1 ± 0.1%; P = 0.001) of CD4+ T-cells (Figure 10A, B), as well TNF-α production (Vehicle: 6.4 ± 0.4%; NE: 3.7 ± 0.7%; P = 0.01) in CD8+ T-cells (Figure 10C, D), These data strongly suggest that sustained elevated levels of splenic NE following chronic SCI could induce T-cell exhaustion and dysfunction of cytokine production.

Bottom Line: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production.The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells.Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Miami Project to Cure Paralysis, Department of Neurosurgery, Miller School of Medicine, University of Miami, Miami, FL 33136, USA. VBracchi@med.miami.edu.

ABSTRACT

Background: Chronic spinal cord injury (SCI) induces immune depression in patients, which contributes to their higher risk of developing infections. While defects in humoral immunity have been reported, complications in T-cell immunity during the chronic phase of SCI have not yet been explored.

Methods: To assess the impact of chronic SCI on peripheral T-cell number and function we used a mouse model of severe spinal cord contusion at thoracic level T9 and performed flow cytometry analysis on the spleen for T-cell markers along with intracellular cytokine staining. Furthermore we identified alterations in sympathetic activity in the spleen of chronic SCI mice by measuring splenic levels of tyrosine hydroxylase (TH) and norepinephrine (NE). To gain insight into the neurogenic mechanism leading to T-cell dysfunction we performed in vitro NE stimulation of T-cells followed by flow cytometry analysis for T-cell exhaustion marker.

Results: Chronic SCI impaired both CD4+ and CD8+ T-cell cytokine production. The observed T-cell dysfunction correlated with increased expression of programmed cell death 1 (PD-1) exhaustion marker on these cells. Blocking PD-1 signaling in vitro restored the CD8+ T-cell functional defect. In addition, we showed that chronic SCI mice had higher levels of splenic NE, which contributed to the T-cell exhaustion phenotype, as PD-1 expression on both CD4+ and CD8+ T-cells was up-regulated following sustained exposure to NE in vitro.

Conclusions: These studies indicate that alteration of sympathetic activity following chronic SCI induces CD8+ T-cell exhaustion, which in turn impairs T-cell function and contributes to immune depression. Inhibition of the exhaustion pathway should be considered as a new therapeutic strategy for chronic SCI-induced immune depression.

Show MeSH
Related in: MedlinePlus