Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

Abstract

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

Figure 1:. Experimental design.

Mice were randomly divided into six groups. Two groups underwent cecal ligation and puncture (CLP) at DO, and the others were sham-operated or not operated. Three days after surgery (D3), S. aureus [SA, 1 × 10⁷ colony forming units (CFU)] or normal saline (NS) was administered intranasally. The control group (Ctrl) mice were not intranasally instilled. Blood, bronchoalveolar lavage fluid (BALF), and peritoneal lavage fluid (PLF) were harvested 24 h after SA injection for a bacteria count assay. The mortality rate in each group was observed over the course of 7 days for survival analysis.

Figure 2:. Mouse survival.

C57BL/6 mice submitted to either CLP or sham surgery received S. aureus (SA) or normal saline (NS) on the third day after surgery (n = 8 mice per group). The mice were monitored for 7 days, and mortality was recorded every 12 h. Kaplan-Meier survival curves, log-rank test (*p < 0.05) compared with Sham+SA mice and SA mice.

Figure 3:. Mouse cecum.

Secondary infection was induced 3 days post-CLP. 24 h after SA infection, mice were sacrificed to collect colon tissues. The representative photos of cecal ligation under different injury hit are shown. SA = S. aureus:NS = normal saline; CLP = cecal ligation and puncture. Scale bar = 1 cm.

Figure 4:. Bacterial counts and representative images of agar plates.

3 days after surgery, mice were intranasally instilled with 1 × 10⁷ CFU of S. aureus (SA) (n > 3 mice per group). Blood, BALF, and PLF were harvested 24 h after SA injection, and bacterial colony counts were determined after 24 h of incubation. Results are expressed as mean ± SEM. One-way ANOVA (Tukey’s post hoc; *p < 0.05; **p < 0.01 ; ***p < 0.001 ; ns = not significant). Please click here to view a larger version of this figure.

Figure 5:. ELISA detecting cytokine secretion.

Secondary infection was induced 3 days post-CLP. Concentrations of IL-1β, IL-6, and TNFα in serum (A) and BALF (B) from mice after SA infection (n > 3 mice per group). Data are presented as the mean ± SEM of three experiments. Oneway ANOVA (Tukey’s post hoc; *p < 0.05; **p < 0.01 ; ****p < 0.0001 ; ns = not significant).

Figure 6:. Representative frequency of neutrophil penetration.

24 h after intranasal infection, mice were sacrificed to obtain BALF (n > 3 mice per group). The percentage of neutrophils (CD11b⁺, GR-1⁺) was quantified by flow cytometry and are shown as means ± SEM of three experiments. One-way ANOVA (Tukey’s post hoc; ****p < 0.0001; ns = not significant).