Therapeutic effect of human ghrelin and growth hormone: Attenuation of immunosuppression in septic aged rats

Abstract

Sepsis is a leading cause of mortality in intensive care units, and is more common in the geriatric population. The control of hyperinflammation has been suggested as a therapeutic approach in sepsis, but to date clinical trials utilizing this strategy have not lead to an effective treatment. In addition to hyperinflammation, patients with sepsis often experience a state of immunosuppression, which serves as an important determinant for increased morbidity and mortality. We previously used aged animals to demonstrate the effectiveness of combined treatment with human ghrelin (Ghr) and human growth hormone (GH) in improving organ injury and survival in septic animals. Here, we hypothesized that combined treatment with Ghr and GH could improve immune function in septic aged animals. Male 24-month-old rats were subjected to cecal ligation and puncture (CLP) for sepsis induction. Human Ghr (80nmol/kg BW) plus GH (50μg/kg BW) or vehicle (normal saline) was administrated subcutaneously at 5h after CLP. The ex vivo production of TNF-α, IL-6 and IL-10 to LPS-stimulation, as well as TNF-α, IL-6, IL-10 and IFN-γ production to anti-CD3/anti-CD28 antibody-stimulation, in splenocytes isolated 20h after CLP, was significantly decreased compared to production of these cytokines in splenocytes from sham animals. The production of cytokines from splenocytes isolated from septic animals that received the combined treatment, however, was significantly higher than from those isolated from vehicle-treated septic animals. Combined treatment prevented the loss of splenic CD4⁺ and CD8⁺ T cells in septic aged rats, and reduced lymphocyte apoptosis. Combined treatment also inhibited an increase in the regulatory T cell (T_reg) population and expression of the immune co-inhibitory molecule PD-1 in the spleens of septic aged rats. In contrast, expression of HLA-DR was increased after combined treatment with Ghr and GH. Based on these findings, we conclude that co-administration of Ghr and GH is a promising therapeutic tool for reversing immunosuppression caused by sepsis in the geriatric population. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Keywords: Aging; Ghrelin; Growth hormone; Immunosuppression; Sepsis.

Figure 1

Cytokine secretion from splenocytes of septic aged rats after LPS-stimulation. Aged rats were subjected to sham operation or CLP with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg GH) treatment at 5 h after CLP. Splenocytes were isolated at 20 h after CLP and further stimulated with LPS (100 ng/ml) for 5 h. Levels of TNF-α (A), IL-6 (B) and IL-10 (C) in the culture supernatant were measured. Data are expressed as mean ± SEM (n = 5–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle-treated septic animals.

Figure 2

Cytokine secretion from splenocytes of septic aged rats after anti-CD3/anti-CD28-antibody stimulation. Aged rats were subjected to sham operation or CLP with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg GH) treatment at 5 h after CLP. Splenocytes were isolated at 20 h after CLP and further stimulated with anti-rat CD3 and anti-rat CD28 antibodies (1 μg/ml each) for 20 h. Levels of TNF-α (A), IL-6 (B), IL-10 (C) and INF-γ (D) in the cell culture supernatant were measured. Data are expressed as mean ± SEM (n = 5–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle-treated septic animals.

Figure 3

Apoptosis of CD4⁺ and CD8⁺ T cells in the spleens of septic aged rats. Aged rats were subjected to sham operation or CLP with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg GH) treatment at 5 h after CLP. Spleens were harvested at 20 h after CLP. Sections of splenic tissues were immunostained for CD4 and CD8 (A). In another group of animals, isolated splenocytes were stained with PE/Cy7-labeled anti-CD4 or APC-labeled anti-CD8 antibodies followed by FITC-labeled TUNEL reaction and analyzed by flow cytometry (B–C). Isotype-matched antibodies were used as negative controls. Data are expressed as mean ± SEM (n = 5–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle-treated septic animals.

Figure 4

T_reg population in the spleens of septic aged rats. Aged rats were subjected sham operation or CLP with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg GH) treatment at 5 h after CLP. Spleens were harvested at 20 h after CLP. Isolated splenocytes were stained for PE/Cy7-labeled anti-CD4 antibody (A and B), followed by staining with PE-labeled anti-FOXP3 antibody (C). The percentage of T_reg cells (CD4⁺ FOXP3⁺) was assessed by flow cytometry (D). Data are expressed as mean ± SEM (n = 5–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle-treated septic animals.

Figure 5

Expression of PD-1 in the spleens of septic aged rats. Aged rats were subjected to sham operation or CLP with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg of GH) treatment at 5 h after CLP. Spleens were harvested at 20 h after CLP. Paraffin sections of splenic tissues were immunostained for PD-1 (A). Positively-staining cells appear brown. Representative images of stained spleen sections are shown. Original magnification 200×. PD-1 levels in spleen tissues were measured by Western blotting (B). Data are expressed as mean ± SEM (n = 4–5 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle-treated septic animals.

Figure 6

Expression of PD-L1 and CTLA4 in the spleens of septic aged rats. Aged rats were subjected to sham or CLP operation and treated with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg of GH) 5 h after CLP. Spleens were harvested at 20 h after CLP. Paraffin sections of splenic tissues were immunostained for PD-L1 and CTLA4. Positively-staining cells appear brown. Representative images of stained spleen sections are shown. Original magnification 200× and 400× (Insert).

Figure 7

Expression of HLA-DR in the spleens of septic aged rats. Aged rats subjected to sham operation or CLP with vehicle (normal saline) or GG (80 nmol/kg of Ghr and 50 μg/kg of GH) treatment at 5 h after CLP. Spleens were harvested at 20 h after CLP. Sections of splenic tissues were immunostained for HLA-DR. Positively-staining cells appear brown. Representative images of stained spleen sections are shown (A). In addition, HLA-DR levels in spleen tissues were measured by Western blotting (B). Data are expressed as mean ± SEM (n = 4–5 per group). * P < 0.05 versus sham and ^#P < 0.05 versus vehicle-treated septic animals.

Figure 8

Summary of findings. Immune function is severely impaired in aged animals after sepsis, which is associated with increased T cell apoptosis, elevation in the T_reg population, upregulation of the immune inhibitory signal, and downregulation of the immune initiating molecule HLA-DR. Combined treatment with human Ghr and GH reverses these changes in septic aged animals to restore immune function.