Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related oligopeptides

Abstract

The marked improvement of several immune-mediated inflammatory diseases during pregnancy has drawn attention to pregnancy hormones as potential therapeutics for such disorders. Low molecular weight fractions derived from the pregnancy hormone human chorionic gonadotrophin (hCG) have remarkable potent immunosuppressive effects in mouse models of diabetes and septic shock. Based on these data we have designed a set of oligopeptides related to the primary structure of hCG and tested these in models of septic shock in mice and rhesus monkeys. We demonstrate that mice exposed to lipopolysaccharide (LPS) and treated subsequently with selected tri-, tetra-, penta- and hepta-meric oligopeptides (i.e. MTR, VVC, MTRV, LQGV, AQGV, VLPALP, VLPALPQ) are protected against fatal LPS-induced septic shock. Moreover, administration of a cocktail of three selected oligopeptides (LQGV, AQGV and VLPALP) improved the pathological features markedly and nearly improved haemodynamic parameters associated with intravenous Escherichia coli-induced septic shock in rhesus monkeys. These data indicate that the designed hCG-related oligopeptides may present a potential treatment for the initial hyperdynamic phase of septic shock in humans.

Fig. 1

Structure of β-human chorionic gonadotrophin (hCG) with loop 2 and the amino acid sequence of loop 2 indicated. Adapted from Lapthorn et al. [16]. Arrows point to the preferential cleavage sites in loop 2 (aa_41–57).

Fig. 2

Survival of mice with lipopolysaccharide (LPS)-induced septic shock after treatment with a single dose of different human chorionic gonadotrophin (hCG)-related oligopeptides. Mice were treated with the following oligopeptides (5 mg/kg body weight i.p.): phosphate-buffered saline (PBS) (red triangle) MTR (aa_41–43; black circle, dashed line), MTRV (aa_41-44; brown circle, dashed line), LQG (aa_45–47; green triangle), LQGV (aa_45–48; blue diamond), AQGV (alanine replaced oligopeptide of LQGV; aa_45–48; orange diamond), VLPALP (aa_48–53; black diamond), VLPALPQ (aa_48–54; black triangle) and VVC (aa_55–57; green circle, dashed line). Mice were treated either 2 h (a) or 24 h (b) after administration of LPS intraperitoneally. Cumulative data are presented from three independent experiments (five to six animals per group/experiment). The survival percentages at 84 h were highly significant (P = 0·000001, two-tailed Fisher's exact test) for peptides LQGV, AQGV and VLPALP at 2 h and for peptides MTR, MTRV, VLPALPQ, VVC and AQGV compared to the PBS group.

Fig. 3

The effect of in vivo treatment of BALB/c mice with the oligopeptides LQGV and AQGV (5 ml/kg) on the in vitro CD3/interleukin (IL)-2 (a) and (b) lipopolysaccharide (LPS)-induced splenocyte proliferation. Both oligopeptides significantly (*P < 0·05) reduced in vitro the capacity of the splenocytes to proliferate upon CD3/IL-2 stimulation. However, splenocytes derived from AQGV peptide-treated mice also showed significantly (*P < 0·05) reduced in vitro proliferative response to LPS after 24 h of stimulation, while no significant differences in in vitro proliferative response of splenocoytes derived from LQGV peptide-treated mice to LPS were found. The results presented are from a single experiment and representative of at least three independent sets of experiments (n = 5).

Fig. 4

Reduced lung pathology treatment with oligopeptide cocktail. The picture shows control monkey Ri 429 (a) and the oligopeptide-treated monkey Ri 459 (a). The marked difference in pulmonary oedema, vascular congestion and haemorrhages is easily visible.

Fig. 6

Decreased severity of septic shock-related microscopic lesions after treatment of rhesus monkeys with a cocktail of three human chorionic gonadotropin (hCG)-related oligopeptides. Microscopic examination of tissues of control monkey Ri8012 (A, C, E) demonstrates marked vascular hyperaemia, vasodilation, multi-focal haemorrhages, marked oedema, mildly increased number of multi-focal lymphoplasmacytic infiltrates and sloughing (necrosis and loss) of epithelium in the lamina propria of the gastric mucosa (A) while the treated monkey Ri11152 (B) displays significantly minimized changes. The large intestinal mucosa in control monkey Ri8012 (C) exhibits moderate vascular dilation, active mucosal congestion, multi-focal areas of extravasated red blood cells (haemorrhage), mild to moderate oedema and mildly increased lymphoplasmacytic infiltrates, while the treated monkey Ri11152 (D) exhibits mild vascular hyperaemia and oedema with few small haemorrhagic foci. The hepatic parenchyma of control monkey Ri8012 (E) shows variable number of multi-focal sinusoidal neutrophils (periportal, random sinusoidal and intravascular), multi-focal hyperaemic blood vessels, multi-focal haemorrhage and oedema, while microscopic hepatic alterations of the treated monkey Ri11152 (F) are restricted to the presence of occasional sinusoidal neutrophils.

Fig. 5

Diminished septic shock-related gross pathological findings after treatment of rhesus monkeys with a cocktail of three human chorionic gonadotrophin (hCG)-related oligopeptides. Escherichia coli-related septic shock was induced in eight rhesus monkeys. Six animals were killed at 8 h in order to evaluate the effect of treatment with hCG-related oligopeptides at the level of histopathology after the acute phase of septic shock relative to phosphate-buffered saline (PBS)-treated control monkeys. A significant difference was observed between tissues from control monkeys (e.g. Ri8012; A, B) and treated monkeys (e.g. Ri7046: C, D; Ri11152: E, F). The gastric mucosa of control monkey Ri8012 (B) exhibits marked oedema, congestion and haemorrhage of the cardia (c) and fundus (f) and multiple petechial haemorrhages in the pylorus (p), while the treated monkeys (e.g. Ri7046; D and Ri11152; E) have milder lesions in the cardia and pylorus. Furthermore, the intestinal wall of the large intestine of control animals (e.g. Ri8012; A) exhibits multi-focal to coalescing areas of haemorrhage and oedema, while treated animal Ri7046 (C) shows milder lesions and treated animal Ri11152 (F) exhibits only scattered petechial haemorrhagic foci.

Fig. 7

Plasma levels of inflammatory cytokines. Escherichia coli infusion of rhesus monkeys was associated with an increase of interleukin (IL)-1β, tumour necrosis factor-α, IL-6 and IL-8. The plasma levels of these proinflammatory cytokines did not differ markedly between the oligopeptide-treated monkeys and the untreated monkeys.