Splenectomy Protects against Sepsis Lethality and Reduces Serum HMGB1 Levels

Animal experiments

Male 6–8 wk old BALB/c mice were purchased from Taconic Farms and allowed to acclimate for 7 days before experiments. All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC). Mice were housed in the animal facility of the Feinstein Institute for Medical Research under standard temperature, light, and dark cycles.

Cecal ligation and puncture (CLP)

Mice were subjected to CLP surgery as described previously (16). Following anesthesia with an i.m. injection of ketamine (75 mg/kg, Fort Dodge) and xylazine (20 mg/kg, Bohringer Ingelheim), a 15 mm midline abdominal incision was made to expose the cecum. After ligation at 5.0 mm from the cecal tip, the cecal stump was punctured once with a 22-gauge needle, and a small amount of stool (1 mm in length) was extruded. The cecum was returned to the abdominal cavity, and the incision was closed with running 6–0 prolene suture. All animals were administered normal saline resuscitation (20 ml/kg of body weight, injected subcutaneously), and a single dose of antibiotics (Primaxin, 0.5 mg/mouse in 200 μl sterile saline, injected subcutaneously, Merck) 15 min after surgery. Survival was monitored for 3 wk. To determine levels of HMGB1 and cytokines in the serum, parallel experiments were conducted and mice were euthanized 24 or 44 h after CLP to collect blood and tissues (livers, kidneys, hearts, small intestine, thymus, and spleens). In some experiments, septic mice were given caspase inhibitor (Z-VAD-FMK) or control peptide (Z-FA-FMK, 0.5 mg/mouse, i.p.) 90 min and 12 h after CLP. Animals were euthanized 24 h after CLP; spleen and thymus were collected for analyses of apoptosis. In survival experiments, caspase inhibitor or control peptide was administered 90 min after CLP and/or splenectomy surgeries, and then every 12 h for a total of 3 days. Survival was monitored for 3 wk.

Splenectomy

When mice were subjected to both CLP and splenectomy surgeries, they received splenectomy immediately before CLP. Mice were anesthetized with ketamine and xylazine as described above. The spleen was identified following a midline laparotomy incision, and removed after appropriate blood vessel ligation. Sham animals underwent laparotomy without removing the spleen.

Blood bacterial counts

Blood bacteria were recovered as previously described (3). In brief, 5 μl of whole blood was diluted with PBS and plated as 0.15 ml aliquots on tryptic soy agar plates. Colony forming units (CFU) were counted after overnight incubation at 37°C and expressed as CFU/ml blood.

Cytokine measurements

Serum HMGB1 concentrations were determined by Western immunoblotting analysis as previously described (2, 3). HMGB1 concentrations were calculated with reference to standard curves generated with purified HMGB1 as described previously (3). Serum levels of TNF, IL-2, IL-4, IL-6, IL-10, IL-12, and IFN-γ were measured using Cytometric Bead Array (BD Biosciences) according to the manufacturer’s instructions.

Analyses of tissue histology and apoptosis

Immediately after euthanasia, tissues were collected and fixed in 10% formalin, and sections were made and stained with H&E for morphologic evaluations.

For apoptosis analyses, TUNEL staining was performed by using ApoAlert DNA fragmentation assay kit (BD Biosciences) and caspase 3 expression was determined by using immunostaining kits from Vector Laboratories. Staining and analyses were performed by blinded observers. Quantifications were performed by counting a representative area of tissue section of each slide.

Statistical analysis

Data are presented as mean ± SEM. Differences between treatment groups were determined by a Student’s t test or one-way ANOVA followed by the least significant difference test or Fisher’s exact test (for survival experiments); p values <0.05 were considered statistically significant.

Splenectomy protects against sepsis lethality, reduces serum HMGB1 levels, prevents liver injury, and maintains a protective Th-1 cytokine response

To investigate the relationship between systemic cytokine release, sepsis lethality, and apoptosis in the spleen, BALB/c mice were subjected to splenectomy or sham surgery immediately before CLP surgery, a standardized murine model of intra-abdominal sepsis (16). We observed that splenectomy significantly improves survival compared with sham surgery (Fig. 1⇓A). Survival of animals was monitored for 3 wk, indicating that splenectomy provides lasting protection against sepsis lethality. To examine the effects of splenectomy on systemic cytokine release, animals were subjected to splenectomy or sham surgery before CLP, and euthanized 24 or 44 h after CLP surgery. We observed that splenectomy significantly reduces serum HMGB1 levels compared with sham-operated controls (Fig. 1⇓B). In addition, tissue histology revealed that CLP sepsis causes liver damage, as demonstrated by significant cellular edema (Fig. 1⇓C). Splenectomy conferred significant protection against liver injury as revealed by near normal liver structure, with some cell edema (Fig. 1⇓C). No apparent differences were observed in tissue sections obtained from kidney and small intestine (data not shown). We also examined whether splenectomy reverses the imbalance of the Th-1 and Th-2 cytokine response seen in sepsis (12). We observed that splenectomy significantly increases serum concentrations of Th-1 cytokines such as IL-2, IL-12, and IFN-γ at 44 h after CLP, but not at 24 h (Fig. 1⇓D). In contrast, splenectomy does not significantly alter serum concentrations of Th-2 cytokines, such as IL-4 and IL-10, as compared with sham-operated controls (Fig. 1⇓D). Taken together, these findings suggest that splenectomy has protective effects in sepsis by decreasing systemic HMGB1 release, maintaining a Th-1-dependent serum cytokine response, and reducing sepsis-induced liver injury.

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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.

Protective effects of caspase inhibitors in sepsis require the spleen

We next performed histological analyses of the spleen and thymus. Mice were subjected to CLP with or without splenectomy, treated with caspase inhibitor or control peptide, and then euthanized 24 h after surgeries. As expected, CLP induces significant apoptosis in the white pulp of the spleen, as measured by terminal nuclear DNA condensation and fragmentation (TUNEL) staining (Fig. 2⇓A) and active caspase 3 expression (data not shown). Previous studies showed that caspases are proteases that activate apoptotic cell death, and administration of agents that inhibit caspase activity improves organ function and increases survival in experimental sepsis (12, 13, 14, 15, 17). Caspase inhibitors fail to protect mice lacking functional T and B lymphocytes, indicating that lymphocytes are critical to the caspase-dependent protective effects (17). To study the role of caspases and splenectomy in sepsis, we administered a broad-spectrum caspase inhibitor, Z-VAD-FMK, or negative control peptide, Z-FA-FMK, to splenectomized or sham-operated mice during CLP sepsis. Consistent with previous studies (11, 13, 17), we found that treatment with Z-VAD-FMK significantly reduces splenic apoptosis as revealed by TUNEL staining (Fig. 2⇓A). CLP also induces significant apoptosis in the cortex of the thymus (Fig. 2⇓B). Administration of Z-VAD-FMK significantly reduces thymic apoptosis in splenectomized septic mice as shown by TUNEL (Fig. 2⇓B) and active caspase 3 staining (data not shown). As expected, extensive apoptosis is present in the thymus of splenectomized mice in a magnitude comparable to sham-operated septic mice with intact spleen (Fig. 2⇓B).

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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.

To further explore the specific role of spleen apoptosis and systemic HMGB1 release, we administered Z-VAD-FMK, or control peptide, Z-FA-FMK, to splenectomized or sham-operated mice during CLP sepsis. Consistent with our previous findings (11), administration of Z-VAD-FMK significantly reduces serum HMGB1 levels as compared with septic mice treated with control peptide (Fig. 2⇑C). In contrast, treatment of splenectomized septic mice with Z-VAD-FMK increases serum HMGB1 levels (Fig. 2⇑C) and fails to protect against sepsis lethality (survival = 40% in control group vs 25% in Z-VAD-FMK treated group, n = 11 mice per group, p = NS). Administration of Z-VAD-FMK to splenectomized, nonseptic mice does not influence serum HMGB1 levels (Fig. 2⇑C). There is no statistical difference in blood bacterial counts between caspase inhibitor or control peptide-treated splenectomized mice or sham-splenectomized controls, suggesting that these effects are not due to changes in bactericidal clearance mechanism (Fig. 2⇑D). Together, these findings indicate that caspase-dependent apoptosis in the spleen, rather than the thymus, is specifically responsible for HMGB1 release in this model of polymicrobial sepsis, and specific prevention of spleen apoptosis protects animals against HMGB1-mediated sepsis lethality.