Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis

Abstract

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in Bcl-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.

Figure 1

Histological GFP-positive cells. Mice were injected i.v. (Upper) or intrathymically (Lower) with 10¹⁰ particles of an adenoviral vector containing an empty cassette or expressing GFP. Tissues were examined 24 h later for GFP fluorescence or by hematoxylin and eosin (H&E) staining. (Magnification: ×100.)

Figure 2

Survival rate of different treatment and nontreatment groups. Survival rate of mice undergoing CLP without treatment (●) was compared with that of animals pretreated intrathymically with 10⁵ particles of an adenoviral vector expressing hIL-10 (Adv/hIL-10) (○) 24 h before CLP and mice pretreated with an equivalent number of adenoviral particles containing an empty cassette (▾). Transgenic mice overexpressing Bcl-2 in T cells (■) as well as sham mice (▿) had a survival of 100%. *, P < 0.05 CLP vs. treatment or sham; †, P < 0.05 Adv/hIL-10 vs. Adv/empty, by Kaplan-Meier, log rank.

Figure 3

Caspase-3-like activity in the thymus of septic mice. Caspase-3-like activity was determined in the thymus from septic mice (n = 10/group) 24 h after CLP and 48 h after intrathymic or i.v. pretreatment. Data from several experiments were combined, and the rate of caspase-3 activity in the thymus of septic mice was normalized to 100%. Statistical comparisons could not be performed between groups of mice treated intrathymically and i.v. because of the effect of a prior surgery (thoracotomy) on subsequent thymic caspase activity. Data were normalized (100%) for values from sham, septic mice [2,329 relative fluorescence intensity (RFI) for intrathymic and 301 RFI for i.v. injections]. *, P < 0.05 CLP vs. treatment or sham; †, P < 0.05 Adv/hIL-10 vs. Adv/empty, by one-way ANOVA and Fischer's least significant difference posthoc test.

Figure 4

In situ TUNEL staining and histological examination of thymi from mice after CLP. Thymi were harvested from mice 24 h after CLP, and tissues were stained with hematoxylin and eosin (H&E), or by 3′ end labeling of apoptotic nuclei (TUNEL) staining as described in Materials and Methods. Increased numbers of cells undergoing apoptosis were seen in mice after CLP. Mice pretreated intrathymically with 10⁵ particles of Adv/empty had similar numbers of apoptotic cells, as determined by TUNEL staining. In mice pretreated with 10⁵ particles of Adv/hIL-10, there was a marked reduction in the numbers of apoptotic cells. In the hematoxylin and eosin-stained sections (see Insets for greater detail), apoptotic cells (fragmented and pyknotic) were seen primarily in the thymus of untreated mice and mice pretreated with Adv/empty. [Magnification: ×100 and ×1,000 for Insets].)

Figure 5

Bcl-2 expression in thymus as determined by Western blot analysis. Bcl-2 levels were examined in the thymus of healthy mice and mice after a CLP. Mice were pretreated intrathymically with either Adv/hIL-10 or Adv/empty at a dose of 10⁵ particles. Bcl-2 levels were determined by Western blot analysis (A and C), and the relative quantities of Bcl-2 were determined by densitometric analysis (B and D). *, P < 0.05 healthy vs. CLP; †, P < 0.05 Adv/hIL-10 vs. Adv/empty or CLP; ‡, P < 0.05 healthy vs. sham. As a positive control for detecting murine Bcl-2, M1 cell lysate (ATCC TIB 192) was used.