Immune suppression reversal of the spleen: a promising strategy for improving the survival rate of sepsis in rats

Abstract

Evidence suggests that immune dysfunction exerts a central role in the morbidity and mortality of sepsis. As the spleen is the largest lymphatic tissue in the body, its influence on immune regulation during sepsis should be explored. In this study, we analysed the immune alterations of the spleen of septic rats and the effects of splenectomy at 6 h, 12 h, and 24 h following caecal ligation and puncture (CLP). Results showed declines in CD4⁺ T cells and elevations in lymphocyte apoptosis, the percentage of Treg cells, and inflammatory cytokine levels (TNF-α, IL-6, and IL-10) in the spleens of CLP-induced septic rats. Moreover, splenectomy improved the survival of septic rats and bacterial clearance from peripheral blood. CLP-induced apoptosis of lymphocytes and the decreased CD4⁺ T cell percentage in the peripheral blood could be reversed in splenectomy-treated rats. Splenectomy greatly decreased the number of white blood cells, lymphocytes, monocytes, neutrophils, and serum concentration of TNF-α and IL-10 after CLP. Moreover, splenectomy alleviated pathologic damage to the liver and lungs and weakened expression of CD163. These novel findings demonstrate that immune disorders of the spleen are important pathogenic factors during the course of severe sepsis. Splenectomy could alleviate apoptosis and reduction of lymphocytes induced by sepsis, and lower the level of inflammation in the body. Reversing the immune suppression of the spleen may be a novel strategy to improve sepsis survival.

Keywords: CD163; Splenectomy; cell apoptosis; immune suppression; sepsis.

Figure 1

Survival rate, blood bacterial counts, spleen index, and pathologic staining of the spleen of septic rats induced by cecal ligation and puncture (CLP). A. Survival was followed for 3 days (n=15 per group). B. The blood bacterial counts were obtained by calculating the number of colony forming units (CFU) per mL of the peripheral blood (n=6 per group). C. Spleen index were calculated by (spleen weight/rat weight) ×10. D. Hematoxylin-eosin and TUNEL stain for spleen at 6 h, 12 h and 24 h after CLP operation (Original magnification, 100×). (*P<0.05, **P<0.01, ***P<0.001 compared to control group).

Figure 2

Changes in the percents of lymphocyte apoptosis and T lymphocyte typing, and expression levels of inflammatory cytokines in spleen of septic rats at 6 h, 12 h and 24 h after CLP operation. A. Flow cytometry for the early and late lymphocyte percents of apoptosis. The P1 gate is used to represent lymphocytes (red dots). B. Flow cytometry for the percents of CD3⁺CD4⁺ T cell, CD3⁺CD8⁺ T cell, and Treg cell; CD3⁺CD4⁺ T cell/CD3⁺CD8⁺ T cell were conducted to evaluate the immune level of spleen (n=6 per group). C. ELISA assay for TNF-α, IL-6, and IL-10 concentrations in the spleen (n=3 per group). (*P<0.05, **P<0.01 compared to control group).

Figure 3

Effects of splenectomy on septic rats. A. Survival was followed for 3 days (n=15 per group). B. The blood bacterial counts were obtained by calculating the number of colony forming units (CFU) per mL of the peripheral blood. C. Flow cytometry for the early and late lymphocyte percents of apoptosis at 6 h, 12 h, and 24 h. D. Routine blood tests for the number of white blood cell, lymphocytes, monocytes, neutrophils and blood platelets in peripheral blood (n=3 per group). E. Flow cytometry for the percents of CD3⁺CD4⁺ T cells, CD3⁺CD8⁺ T cells and CD3⁺CD4⁺ T cells/CD3⁺CD8⁺ T cells were conducted to evaluate the immune level (n=6 per group). F. ELISA assay for serum TNF-α and IL-10 (n=3 per group). (*P<0.05, **P<0.01, ***P<0.001 compared to control group; #P<0.05, ##P<0.01, ###P<0.001 compared to CLP group).

Figure 4

Effects of splenectomy on liver and lung in septic rats. Hematoxylin-eosin stain (A) and the expression of CD163 by immumohistochemical staining (B) on liver and lung were shown at 24 h after CLP operation (Original magnification, ×200). (C) The statistical results are expressed as number of positive cells per 1000 cells in the liver or lung. (***P<0.001 compared to control group; ##P<0.01 compared to CLP group, n=4 per group).