Splenectomy protects against sepsis lethality and reduces serum HMGB1 levels

Abstract

High mobility group box 1 (HMGB1) is a critical mediator of lethal sepsis. Previously, we showed that apoptotic cells can activate macrophages to release HMGB1. During sepsis, apoptosis occurs primarily in lymphoid organs, including the spleen and thymus. Currently, it is unclear whether this accelerated lymphoid organ apoptosis contributes to systemic release of HMGB1 in sepsis. In this study, we report that splenectomy significantly reduces systemic HMGB1 release and improves survival in mice with polymicrobial sepsis. Treatment with a broad-spectrum caspase inhibitor reduces systemic lymphocyte apoptosis, suppresses circulating HMGB1 concentrations, and improves survival during polymicrobial sepsis, but fails to protect septic mice following splenectomy. These findings indicate that apoptosis in the spleen is essential to the pathogenesis of HMGB1-mediated sepsis lethality.

Figures

FIGURE 1

Splenectomy (SPX) improves survival, reduces serum HMGB1 levels, and ameliorates liver injury in septic mice. A, BALB/c mice (male, 20–25 gm) underwent splenectomy or sham surgery before CLP. Survival was monitored for 3 wk. Data are shown as percent of animals surviving (n ≥ 35 per group). *, p < 0.01 vs CLP group as tested by Fisher’s exact test. B, BALB/c mice underwent splenectomy or sham surgery before CLP. Animals were euthanized at 44 h after surgeries and sera were collected for determination of HMGB1 levels by Western blot analysis, as described previously (3). Data are presented as mean ± SEM (n = 8–11 per group) *, p < 0.05 vs CLP group. C, BALB/c mice underwent splenectomy or sham surgery before CLP and were euthanized at 44 h after surgeries. Selected organs were collected. Tissue sections of livers from normal and septic mice were prepared by using a standard formalin-fixed, paraffin-embedded procedure. Tissues were cut in 4-μm slides, mounted on glass slides, and stained with H&E. Left, Normal liver shows a central vein (arrow) surrounded by normal hepatocytes and sinusoids. Middle, CLP causes cellular damage, as demonstrated by significant cell edema. Right, Splenectomy prevents sepsis-induced damage and maintains near-normal hepatocytes. Data are representative of 9–14 mice per group. Magnifications, ×100. D, BALB/c mice underwent splenectomy or sham surgery and CLP. Mice were euthanized at either 24 or 44 h after surgeries. Serum levels of Th-1 cytokines IL-12, IFN-γ, and IL-2, Th-2 cytokines IL-4 and IL-10, and other cytokines including TNF and IL-6, were measured by using cytometric bead array according to the manufacturer’s instructions (see Materials and Methods). Data are mean ± SEM (n = 12–14 per group). *, p < 0.05 vs CLP group.

FIGURE 2

Caspase inhibitor Z-VAD-FMK reduces thymic apoptosis but fails to reduce serum HMGB1 levels in splenectomized, septic mice. Male BALB/c mice underwent CLP surgery, or splenectomy (SPX) plus CLP, and were treated with Z-VAD-FMK or control peptide at 0.5 mg/mouse (injected i.p.) at 90 min and 12 h after surgeries. Mice were euthanized 24 h after surgeries for serum and tissue measurements. Tissue sections of spleen and thymus were prepared for staining of apoptosis markers. A, TUNEL staining of spleen. Normal spleen shows white and red pulp areas (arrows indicate white pulp) with minimal apoptosis. CLP induces significant apoptosis with most TUNEL positive cells in the white pulp. Treatment with Z-VAD-FMK significantly reduces apoptosis. Magnifications, ×100. B, TUNEL staining of thymus. Compared with normal mice, CLP induces significant cortical apoptosis in the thymus. Treatment with Z-VAD-FMK reduces apoptosis in CLP and CLP plus splenectomized mice. Splenectomy does not reduce thymic apoptosis in CLP mice (n = 9–11 mice per group). Magnifications, ×100. C, Serum HMGB1 levels at 24 h after CLP and/or splenectomy surgeries, or splenectomy plus Z-VAD-FMK (n = 5–16 mice per group) *, p < 0.05 vs CLP group. D, Blood bacterial counts at 24 h after surgeries (n = 8–16 mice per group), p = NS.

FIGURE 3

Spleen apoptosis is a specific determinant of HMGB1-mediated sepsis lethality. Sepsis causes extensive apoptosis in peripheral lymphoid organs. Phagocytosis of apoptotic cells by macrophages can induce HMGB1 release. HMGB1, a proinflammatory mediator, causes tissue injury and death in sepsis. Caspase inhibitor Z-VAD-FMK inhibits lymphocyte apoptosis in both spleen and thymus. Apoptosis in the spleen, rather than in the thymus, specifically increases systemic HMGB1 release and accelerates sepsis lethality. Prevention of apoptosis in the spleen via splenectomy reduces systemic HMGB1 release and protects against polymicrobial sepsis lethality.