Model of polymicrobial peritonitis that induces the proinflammatory and immunosuppressive phases of sepsis

Abstract

Severe sepsis is associated with early release of inflammatory mediators that contribute to the morbidity and mortality observed during the first stages of this syndrome. Although sepsis is a deadly, acute disease, high mortality rates have been observed in patients displaying evidence of sepsis-induced immune deactivation. Although the contribution of experimental models to the knowledge of pathophysiological and therapeutic aspects of human sepsis is undeniable, most of the current studies using animal models have focused on the acute, proinflammatory phase. We developed a murine model that reproduces the early acute phases but also the long-term consequences of human sepsis. We induced polymicrobial acute peritonitis (AP) by establishing a surgical connection between the cecum and the peritoneum, allowing the exit of intestinal bacteria. Using this model, we observed an acute phase with high mortality, leukopenia, increased interleukin-6 levels, bacteremia, and neutrophil activation. A peak of leukocytosis on day 9 or 10 revealed the persistence of the infection within the lung and liver, with inflammatory hepatic damage being shown by histological examination. Long-term (20 days) derangements in both innate and adaptive immune responses were found, as demonstrated by impaired systemic tumor necrosis factor alpha production in response to an inflammatory stimulus; a decreased primary humoral immune response and T cell proliferation, associated with an increased number of myeloid suppressor cells (Gr-1(+) CD11b(+)) in the spleen; and a low clearance capacity. This model provides a good approach to attempt novel therapeutic interventions directed to augmenting host immunity during late sepsis.

FIG. 1.

(A) Surgical induction of AP. Representative photographs of the ceca of mice with the tube (arrowheads) inserted through the cecum wall to cause the acute peritonitis (left) or fixed without penetrating the cecum wall (right). (B) Percent survival after AP induction (n = 24 per group; *, P < 0.05 versus sham group).

FIG. 2.

Absolute leukocyte counts present in peripheral blood (A, B, and C) and peritoneal infiltrations (D and E) at different times after AP induction. (A) Lymphocytes; (B) neutrophils; (C) monocytes (for panels A to C, n = 6 to 30 animals for each time point, by group; *, P < 0.05 versus sham group); (D) neutrophils; (E) monocytes/macrophages (Mφ). The horizontal lines represent the values observed in healthy untreated mice (reference value) (n = 6 to 30 animals for each time, by group; *, P < 0.05 versus sham group; †, P < 0.05 versus reference value).

FIG. 3.

Presence of TNF-α, IL-6, and IL-10 in serum at different time points (A) and in peritoneal fluid on day 1 (B) after AP induction. Serum and peritoneal fluid specimens were obtained from sham and AP mice at different times after surgery. TNF-α was determined using a biological test, and the results are expressed as the 50% lethal dose (LD₅₀), as described in Materials and Methods. IL-10 and IL-6 levels were determined by ELISA (n = 12; *, P < 0.05 versus sham group).

FIG. 4.

Functional state of neutrophils on day 1 after AP induction. (A) The MFI of the CD11b activation marker was measured in peripheral and peritoneal neutrophils (Ly-6G cells) by flow cytometry. The horizontal line represents the MFI value for untreated healthy mice (n = 9 mice per group; *, P < 0.05 versus sham group). (B) The adhesive properties of neutrophils were evaluated by measuring in vivo the percentage of cells within lung vessels that were positive for the myeloid marker Ly-6G, as described in Materials and Methods (n = 6 mice per group; *, P < 0.05 versus sham group). Representative dot plots are shown. (C) Representative photographs (May-Grünwald-Giemsa staining) showing toxic alterations on peripheral neutrophils from AP animals.

FIG. 5.

Percentage of positive bacterial cultures and numbers of CFU in peripheral blood, peritoneal fluid, lung, and liver after AP induction. (A) Peripheral blood and peritoneal fluid were collected from AP mice and plated on MacConkey agar (n = 20; n.d., not determined); (B) numbers of CFU/ml obtained on MacConkey agar; (C) the lung and liver were collected and processed as indicated in Materials and Methods and plated on MacConkey or LB agar (n = 13). The table shows the numbers of CFU per organ.

FIG. 6.

Analysis of different immunological parameters at 9 and 20 days after AP induction. (A) Systemic TNF-α production in response to an inflammatory stimulus. Sham and AP mice were injected with 5 μg of LPS, and serum was obtained 1.5 h later. TNF-α levels were determined using the L929 biological method, as described in Materials and Methods (n = 10 per group; *, P < 0.05 versus sham group). (B) Primary humoral immune response. Mice were immunized with SRBCs on day 9 or 20 postsurgery, and the levels of antibodies against SRBCs in the sera of sham and AP mice were measured 7 days later using the hemagglutination assay. Twofold serial dilutions of serum were performed, and the inverse of the maximal dilution where the antigen-antibody complex was observed is provided (n = 12 per group; *, P < 0.05 versus sham group). (C) T cell proliferation. Splenocytes were obtained from sham and AP mice, and 0.2 × 10⁵ lymphocytes were seeded in a 96-well plate in triplicate in the presence of concanavalin A (5 μg/ml). After 72 h, cells were stained using the MTT assay, as described in Materials and Methods, and the resultant absorbance at 550 nm was measured (n = 6 per group; *, P < 0.05 versus sham group). (D) Percentage of Gr-1⁺ CD11b⁺ cells in spleens of sham and AP mice determined by flow cytometry (n = 12 per group; *, P < 0.05 versus sham group). (E) Clearance capacity was determined by inoculating bacteria in the peritoneum and measuring the remaining numbers of CFU in peritoneal lavage fluid specimens 4 h later using MacConkey agar (n = 9 per group; *, P < 0.05 versus sham group).

FIG. 7.

Histological examination in AP mice (hematoxylin-eosin staining). (A) Day 1 after AP, intra-alveolar lung hemorrhage. Magnification, ×250. (Inset) Destroyed alveolar septa. Magnification, ×400. (B) Nine days after AP, focus of hepatic necrosis surrounded by abundant leukocyte infiltration. Magnification, ×250. (Inset) Mixed inflammatory infiltrate with neutrophils and lymphocytes. Magnification, ×1,000. (C) Twenty days after AP, lung with focus of perivascular inflammatory infiltrate. Magnification, ×250. (Inset) A predominantly lymphocytic infiltrate. Magnification, ×400. (D) Twenty days after AP, focus of regenerated hepatocytes with chronic inflammatory infiltrate. Magnification, ×250). (Inset) Predominantly lymphocytic infiltrate. Magnification, ×1,000.