LL-37 protects rats against lethal sepsis caused by gram-negative bacteria

Abstract

We investigated the efficacy of LL-37, the C-terminal part of the only cathelicidin in humans identified to date (termed human cationic antimicrobial protein), in three experimental rat models of gram-negative sepsis. Adult male Wistar rats (i) were given an intraperitoneal injection of 1 mg Escherichia coli 0111:B4 LPS, (ii) were given 2 x 10(10) CFU of Escherichia coli ATCC 25922, or (iii) had intra-abdominal sepsis induced via cecal ligation and puncture. For each model, all animals were randomized to receive intravenously isotonic sodium chloride solution, 1-mg/kg LL-37, 1-mg/kg polymyxin B, 20-mg/kg imipenem, or 60-mg/kg piperacillin. Lethality; growth of bacteria in blood, peritoneum, spleen, liver, and mesenteric lymph nodes; and endotoxin and tumor necrosis factor alpha (TNF-alpha) concentrations in plasma were evaluated. All compounds reduced lethality compared to levels in controls. Endotoxin and TNF-alpha plasma levels were significantly higher in conventional antibiotic-treated rats than in LL-37- and polymyxin B-treated animals. All drugs tested significantly reduced bacterial growth compared to saline treatment. No statistically significant differences between LL-37 and polymyxin B were noted for antimicrobial and antiendotoxin activities. LL-37 and imipenem proved to be the most effective treatments in reducing all variables measured. Due to its multifunctional properties, LL-37 may become an important future consideration for the treatment of sepsis.

FIG. 1.

Effect of LL-37, polymyxin B (POL-B), imipenem (IMP), and piperacillin (PIP) on LPS-induced production of TNF-α (Α) and nitric oxide (B). RAW 264.7 cells were stimulated with 100-ng/ml LPS in the absence or presence of the indicated amounts of each drug. TNF-α (Α) and NO (B) were assayed, respectively, in the cell-free medium of cells incubated for 6 and 24 h. The concentrations of TNF-α and NΟ in untreated cells were 61.49 ± 7.58 pg/ml and 6.15 ± 1.87 μM, respectively. Data are presented as the means of at least three experiments ± SD. Statistical analysis was performed according to Student's t test by comparing samples treated with each drug with control cells stimulated with LPS only. A calculated P value of <0.05 (asterisk) was considered significant.

FIG. 2.

Plasma levels of IL-6 (top), endotoxin (middle), and TNF-α (bottom) after bacterial challenge. For all three parameters, LL-37 and polymyxin B levels were significantly different (P < 0.05) compared to imipenem and piperacillin levels after 2 h.

FIG. 3.

Plasma levels of IL-6 (top), endotoxin (middle), and TNF-α (bottom) after a surgical procedure. For all three parameters, LL-37 and polymyxin B levels were significantly different (P < 0.05) compared to imipenem and piperacillin levels after 2 h.