Characterization and modulation of the immunosuppressive phase of sepsis

Abstract

Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had approximately 40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-gamma) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-gamma production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the host's immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.

FIG. 1.

Evaluation of immune effector cells from spleens harvested from sham-operated mice 1 day after CLP (CLP 1d), 4 days after CLP (CLP 4d), and 7 days after CLP (CLP 7d). (A) NK cells returned to sham-operated levels by 4 days following CLP and had significantly increased by 7 days; dendritic cells remained depleted at 4 days but had significantly increased by 7 days. Neutrophils had significantly increased at both 4 and 7 days following CLP. (B) CD4⁺ lymphocytes had significantly decreased by 1 day and remained depleted through 7 days compared to sham-operated animals, while CD8⁺ lymphocyte counts were significantly depleted at 1 and 7 days post-CLP. (C) CD20⁺ B lymphocytes showed continuous loss over the 7-day period. n = 10 per group (except n = 6 for neutrophils). The error bars indicate standard deviations.

FIG. 2.

Splenocytes from animals harvested and stimulated just prior to secondary pneumonia infection at 4 days (96 h after CLP) revealed significant decreases in IFN-γ secretion compared to splenocytes from sham-operated animals or animals 7 days post-CLP. *, P < 0.01; n = 6 per group. The error bars indicate standard deviations.

FIG. 3.

(A) Animals that underwent secondary pneumonia infection 4 days after CLP showed increasing mortality with increasing doses of P. aeruginosa (Pa) (0.3 A₆₀₀ = 5 × 10⁸ CFU/ml, 0.5 A₆₀₀ = 1 × 10⁹ CFU/ml, and 0.7 A₆₀₀ = 3 × 10⁹ CFU/ml; P < 0.006). (B) Animals that underwent secondary pneumonia at 7 days post-CLP showed no significant difference in survival with increasing doses of bacteria. McF, McFarland standard.

FIG. 4.

Animals 7 days post-CLP and under IFN-γ blockade at the time of secondary injury showed increased mortality compared to control animals. P < 0.028; n = 9 per group. Pseudo, P. aeruginosa.

FIG. 5.

Following septic injury, there is significant loss of immune cells by apoptosis. In animals that underwent CLP alone, analysis of splenic T-cell apoptosis at 4 days revealed significant increases in both caspase 3 and TUNEL. CLP followed by the induction of pneumonia at 4 days (0.5 McFarland standard) had an additive effect, as these animals, when evaluated on day 5 (16 h after the induction of pneumonia), had even greater levels of apoptosis. By 7 days post-CLP, there was a reduction of T-cell apoptosis. Similar to 4-day animals, double-injury animals at 7 days had an increase in T-cell apoptosis compared to their single-injury counterparts. Double-injury animals at 7 days compared to 4 days had significantly decreased levels of T-cell apoptosis. Analysis of B-cell apoptosis from spleens at 4 days postinjury revealed significant increases in apoptosis in animals that underwent CLP alone. As seen in the T cells, CLP followed by the induction of pneumonia at both 4 and 7 days had an additive effect upon injury-related cell death. n = 6 per group. The error bars indicate standard deviations.

FIG. 6.

Following a second septic insult, evaluation of plasma cytokines revealed differences in cytokine levels that were dependent on the timing of the secondary injury. Animals infected with P. aeruginosa pneumonia at 4 days post-CLP had very low levels of IL-1α, IL-6, IFN-γ, and G-CSF and higher levels of the anti-inflammatory cytokine IL-10. Animals that were infected with pneumonia 7 days post-CLP had significant increases in IL-1α, IL-6, and G-CSF compared to animals that underwent pneumonia infection at 4 days. Seven-day animals also had no increase in IL-10 16 h after the induction of pneumonia. n = 6 per group. The error bars indicate standard deviations. ns, not significant.

FIG. 7.

BAL fluid and blood cultures showed increased growth in animals that underwent secondary pneumonia infection 4 days post-CLP compared to 7-day double-injury or single-injury animals. *, P < 0.001. n = 10 per group. The error bars indicate standard deviations.

FIG. 8.

(A) Light microscopic examination was performed on lung specimens harvested 18 h following the induction of pneumonia. Animals infected with pneumonia 4 days following CLP showed increased consolidation of tissue with extensive polymorphonuclear and mononuclear cell infiltration compared to animals infected with pneumonia 7 days post-CLP. (B) Evaluation of light microscopy by a pathologist blinded to sample identity revealed significantly decreased scores for infiltration and consolidation in animals infected with pneumonia 7 days after CLP. P < 0.04; n = 6 per group. The error bars indicate standard deviations.

FIG. 9.

(A) Treatment with AS101 following CLP significantly improved survival after secondary injury; P < 0.017; n = 17 per group. (B) Treatment with anti-IL-10 antibody following CLP improved survival after secondary injury similar to that seen in animals treated with AS101. P < 0.011; n = 15 per group.

FIG. 10.

(A) Blood collected 18 h following the induction of pneumonia showed significantly decreased plasma IL-10 and significantly increased IL-1α and IL-12p40 levels in animals treated with AS101. *, P < 0.003. (B) Splenocytes from animals treated with AS101 showed significant increases in IFN-γ production compared to controls 4 days post-CLP; *, P < 0.001. n = 6 per group. The error bars indicate standard deviations.

FIG. 11.

BAL fluid and blood cultures showed decreased growth with AS101 treatment versus vehicle. *, P < 0.03. n = 6 per group. The error bars indicate standard deviations.

FIG. 12.

(A) Light microscopic examination was performed on lung specimens harvested 18 h following the induction of pneumonia. Control animals showed increased consolidation of tissue with extensive polymorphonuclear and mononuclear cell infiltration. (B) Evaluation of light microscopy by a pathologist blinded to sample identity revealed significantly decreased scores for infiltration and consolidation in animals treated with AS101. *, P < 0.05; n = 6 per group. The error bars indicate standard deviations.