Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis - PubMed (original) (raw)

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

C Oberholzer et al. Proc Natl Acad Sci U S A. 2001.

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.

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Figures

Figure 1

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

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

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

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

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.

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