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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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Chronic spinal cord injury (SCI) increases the sympathetic activity in the spleen. (A) Western blot quantification of tyrosine hydroxylase (TH) in the spleens of uninjured mice (CT) and chronic SCI mice (SCI). Data are normalized to the expression of β-actin. Bar graph represents the mean ± SEM of TH expression in the spleen protein extract and are expressed as percentage of CT. n = 4 mice/group. Data represents two independent experiments. *P < 0.05, two-tailed Student’s t-test. (B) Bar graph represents the mean ± SEM of NE concentration (ng/mg) in the spleen homogenates from CT and SCI mice. n = 5 mice/group. *P < 0.05, one-tailed Student’s t-test.
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Figure 8: Chronic spinal cord injury (SCI) increases the sympathetic activity in the spleen. (A) Western blot quantification of tyrosine hydroxylase (TH) in the spleens of uninjured mice (CT) and chronic SCI mice (SCI). Data are normalized to the expression of β-actin. Bar graph represents the mean ± SEM of TH expression in the spleen protein extract and are expressed as percentage of CT. n = 4 mice/group. Data represents two independent experiments. *P < 0.05, two-tailed Student’s t-test. (B) Bar graph represents the mean ± SEM of NE concentration (ng/mg) in the spleen homogenates from CT and SCI mice. n = 5 mice/group. *P < 0.05, one-tailed Student’s t-test.

Mentions: To understand how chronic SCI induces T-cell exhaustion, we investigated whether the activity of the sympathetic nervous system is altered by SCI. As a surrogate marker of SNS activity we measured the protein expression level of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine (CA) synthesis, in the spleen extract from both control and SCI mice. Compared to uninjured control, TH expression levels in the spleen of injured mice was significantly elevated (Figure 8A) and consistent with significantly higher NE concentrations in the spleen of those injured mice (uninjured: 0.26 ± 0.03 ng NE/mg spleen; chronic SCI: 0.38 ± 0.05 ng NE/mg spleen, P = 0.04) (Figure 8B). These results led us to hypothesize that higher sustained splenic NE levels in injured mice may contribute to T-cell exhaustion. To test this hypothesis, we sought to incubate enriched T-cells from naive mice with 10 μM NE over several days and assess whether these cells up-regulated PD-1. Since prolong exposure to NE could be toxic to the cells, we first determined cell viability. Naïve enriched T-cells were incubated either with NE or vehicle for one, two and three days and cell viability was measured by flow cytometry using 7-AAD. As shown in Figure 9, after three days in culture with NE, T-cell viability was very low, as reflected by a low percentage of 7-AAD− live cells (Vehicle: 83.7 ± 3.1%; NE: 11.1 ± 7.7%; P = 0.0009) (Figure 9A, B). Therefore, we determined the number of PD-1 expressing T-cell following one and two days of NE stimulation. As shown in Figure 9C-F, one day of NE stimulation significantly increased PD-1+CD8+ T-cell number compared to vehicle-treated cells (Vehicle: 565 ± 46; NE: 985 ± 75; P = 0.004), when the cell numbers of both CD4+PD-1+ T-cells (Vehicle: 5,283 ± 439; NE: 6,747 ± 135; P = 0.02) and CD8+PD-1+ T-cells (Vehicle: 747 ± 72; NE: 1,031 ± 94; P = 0.04) were upregulated after two days of continuous NE stimulation.


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)

Chronic spinal cord injury (SCI) increases the sympathetic activity in the spleen. (A) Western blot quantification of tyrosine hydroxylase (TH) in the spleens of uninjured mice (CT) and chronic SCI mice (SCI). Data are normalized to the expression of β-actin. Bar graph represents the mean ± SEM of TH expression in the spleen protein extract and are expressed as percentage of CT. n = 4 mice/group. Data represents two independent experiments. *P < 0.05, two-tailed Student’s t-test. (B) Bar graph represents the mean ± SEM of NE concentration (ng/mg) in the spleen homogenates from CT and SCI mice. n = 5 mice/group. *P < 0.05, one-tailed Student’s t-test.
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Figure 8: Chronic spinal cord injury (SCI) increases the sympathetic activity in the spleen. (A) Western blot quantification of tyrosine hydroxylase (TH) in the spleens of uninjured mice (CT) and chronic SCI mice (SCI). Data are normalized to the expression of β-actin. Bar graph represents the mean ± SEM of TH expression in the spleen protein extract and are expressed as percentage of CT. n = 4 mice/group. Data represents two independent experiments. *P < 0.05, two-tailed Student’s t-test. (B) Bar graph represents the mean ± SEM of NE concentration (ng/mg) in the spleen homogenates from CT and SCI mice. n = 5 mice/group. *P < 0.05, one-tailed Student’s t-test.
Mentions: To understand how chronic SCI induces T-cell exhaustion, we investigated whether the activity of the sympathetic nervous system is altered by SCI. As a surrogate marker of SNS activity we measured the protein expression level of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine (CA) synthesis, in the spleen extract from both control and SCI mice. Compared to uninjured control, TH expression levels in the spleen of injured mice was significantly elevated (Figure 8A) and consistent with significantly higher NE concentrations in the spleen of those injured mice (uninjured: 0.26 ± 0.03 ng NE/mg spleen; chronic SCI: 0.38 ± 0.05 ng NE/mg spleen, P = 0.04) (Figure 8B). These results led us to hypothesize that higher sustained splenic NE levels in injured mice may contribute to T-cell exhaustion. To test this hypothesis, we sought to incubate enriched T-cells from naive mice with 10 μM NE over several days and assess whether these cells up-regulated PD-1. Since prolong exposure to NE could be toxic to the cells, we first determined cell viability. Naïve enriched T-cells were incubated either with NE or vehicle for one, two and three days and cell viability was measured by flow cytometry using 7-AAD. As shown in Figure 9, after three days in culture with NE, T-cell viability was very low, as reflected by a low percentage of 7-AAD− live cells (Vehicle: 83.7 ± 3.1%; NE: 11.1 ± 7.7%; P = 0.0009) (Figure 9A, B). Therefore, we determined the number of PD-1 expressing T-cell following one and two days of NE stimulation. As shown in Figure 9C-F, one day of NE stimulation significantly increased PD-1+CD8+ T-cell number compared to vehicle-treated cells (Vehicle: 565 ± 46; NE: 985 ± 75; P = 0.004), when the cell numbers of both CD4+PD-1+ T-cells (Vehicle: 5,283 ± 439; NE: 6,747 ± 135; P = 0.02) and CD8+PD-1+ T-cells (Vehicle: 747 ± 72; NE: 1,031 ± 94; P = 0.04) were upregulated after two days of continuous NE stimulation.

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