Bacterial load plays a crucial role for survival in experimental peritonitis and modulates immunoparalysis of monocytes

Introduction

Peritonitis is the prototype of polymicrobial sepsis with kinetics of bacterial growth differing from those of other infections leading to sepsis. The effect of the extent of leaking of gut content in the response of the host was studied.

Methods

A total of 21 rabbits were studied divided into two groups; A: high-load peritonitis; and B: normal load peritonitis. After a midline abdominal incision, the ileocecal calve was ligated. Three holes were performed in the cecum wall of group A followed by masturbation to drain cecal content in the peritoneal cavity. Two holes without masturbation were performed in group B. After closure of the abdominal cavity, blood was sampled at 2, 4, 24 and 48 hours. Peripheral blood mononuclear cells (PBMCs) were isolated and stimulated in microplate wells with 10 ng/ml LPS. Concentrations of TNFα were estimated in supernatants by a bioassay in L929 fibrosarcoma cell line. In parallel, monocytes were separated from lymphocytes by plastic adherence. Apoptosis was estimated after staining with ANNEXIN-V and PI and flow cytometric analysis. Tissue bacterial growth was estimated after death.

Results

Mortality after 14 days was 84.6% in group A and 62.5% in group B (log-rank: 3.83, P = 0.050). Mean respective rates of apoptosis of lymhocytes of groups A and B were 32.2 and 44.5% at 2 hours; 33.5 and 51.9% at 4 hours (P = 0.028); 35.6 and 39.1% at 24 hours; and 28.5 and 43.7% at 48 hours (P = 0.029). Mean respective rates of apoptosis of monocytes of groups A and B were 48.2 and 64.1% at 2 hours (P = 0.036); 57.9 and 66.3% at 4 hours; 47.3 and 69.9% at 24 hours (P = 0.041); and 60.5 and 73.5% at 48 hours. Respective TNFα released ex vivo from PBMCs isolated at 24 hours from groups A and B after LPS stimulation was 2,579.1 and 31.3 pg/ml (P = 0.048). Mean respective log₁₀ of enterobacteriaceae of groups A and B were 6.52 and 1.39 cfu/ml in liver (P = 0.012); 7.79 and 1.43 cfu/ml in spleen (P = 0.012); and 7.98 and 1.79 cfu/ml in the right kidney (P = 0.012).

Conclusions

Experimental peritonitis with enormous bacterial leaking from the gut is accompanied by reduced survival, increased tissue bacterial growth and reduced apoptosis of lymphocytes and monocytes. Peritonitis with low bacterial leaking is characterized by immunoparalysis of PBMCs. These results may project in the efficacy of immunotherapy in abdominal sepsis.