Recombinant Fasciola hepatica Fatty Acid Binding Protein as a Novel Anti-Inflammatory Biotherapeutic Drug in an Acute Gram-Negative Nonhuman Primate Sepsis Model

Abstract

Due to their phylogenetic proximity to humans, nonhuman primates (NHPs) are considered an adequate choice for a basic and preclinical model of sepsis. Gram-negative bacteria are the primary causative of sepsis. During infection, bacteria continuously release the potent toxin lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to death. Our previous research has demonstrated in vitro and in vivo using a mouse model of septic shock that Fh15, a recombinant variant of the Fasciola hepatica fatty acid binding protein, acts as an antagonist of Toll-like receptor 4 (TLR4) suppressing the LPS-induced proinflammatory cytokine storm. The present communication is a proof-of concept study aimed to demonstrate that a low-dose of Fh15 suppresses the cytokine storm and other inflammatory markers during the early phase of sepsis induced in rhesus macaques by intravenous (i.v.) infusion with lethal doses of live Escherichia coli. Fh15 was administered as an isotonic infusion 30 min prior to the bacterial infusion. Among the novel findings reported in this communication, Fh15 (i) significantly prevented bacteremia, suppressed LPS levels in plasma, and the production of C-reactive protein and procalcitonin, which are key signatures of inflammation and bacterial infection, respectively; (ii) reduced the production of proinflammatory cytokines; and (iii) increased innate immune cell populations in blood, which suggests a role in promoting a prolonged steady state in rhesus macaques even in the presence of inflammatory stimuli. This report is the first to demonstrate that a F. hepatica-derived molecule possesses potential as an anti-inflammatory drug against sepsis in an NHP model. IMPORTANCE Sepsis caused by Gram-negative bacteria affects 1.7 million adults annually in the United States and is one of the most important causes of death at intensive care units. Although the effective use of antibiotics has resulted in improved prognosis of sepsis, the pathological and deathly effects have been attributed to the persistent inflammatory cascade. There is a present need to develop anti-inflammatory agents that can suppress or neutralize the inflammatory responses and prevent the lethal consequences of sepsis. We demonstrated here that a small molecule of 14.5 kDa can suppress the bacteremia, endotoxemia, and many other inflammatory markers in an acute Gram-negative sepsis rhesus macaque model. These results reinforce the notion that Fh15 constitutes an excellent candidate for drug development against sepsis.

Keywords: Fasciola hepatica; cytokines; fatty acid binding protein; rhesus macaques; sepsis.

FIG 1

Levels of antigenemia in plasma samples from rhesus macaques treated with Fh15. A homemade sandwich ELISA was optimized to detect circulating Fh15 in plasma samples. The assay uses a rabbit anti-Fh15 IgG antibody as the capturing antibody and a rabbit anti-Fh15-IgG conjugate with HRP as the detecting antibody. Plasma samples were collected from rhesus macaques at different time points, as follows: 0 h (baseline), 30 min, 2 h, 4 h, 6 h, and 8 h during the experiment. The group termed Fh15 comprised monkeys (n = 3) that received only the i.v. infusion with 15 mg Fh15. The group termed Fh15-E. coli comprised animals (n = 3) that received the i.v. infusion containing 12 mg Fh15 followed by the E. coli infection (live E. coli 10¹⁰ CFU/kg body wt.). The group termed Fh15-Fh15-E. coli comprised animals from the Fh15 group that 3 months later received a second i.v. infusion with 12 mg Fh15 followed by the E. coli infection.

FIG 2

Fh15 decreases bacteremia, endotoxemia, C-reactive protein, and procalcitonin in rhesus macaques during an acute lethal sepsis. Rhesus macaques were allotted into three experimental groups (n = 3) each. The group termed E. coli is the control group comprised by monkeys that received only a lethal i.v. infusion with live E. coli 10¹⁰ CFU/kg of body wt. The group termed Fh15-E. coli comprised monkeys (n = 3) that received the i.v. infusion containing 12 mg Fh15 followed by the E. coli infection. The group termed Fh15-Fh15-E. coli comprised animals from a group that had received a primary i.v. infusion with 12 mg Fh15. Three months later, these monkeys received a second 12-mg Fh15 treatment followed by the E. coli infection. (A) The graphs represent the levels of viable bacteria for each single monkey of each experimental group as well as the comparison among the significant reductions in bacteremia showed by Fh15-E. coli and Fh15-Fh15-E. coli compared with the E. coli control group (***, P = 0.0001). Represents the levels of plasma endotoxin (LPS) (***, P < 0.0001) (B), C-reactive protein (***, P = 0.004) (C), and procalcitonin (****, P < 0.0001) (D). Statistical significance between control group and experimental groups was determining by ANOVA or Student’s t test using GraphPad Prism 8. For all tests, a P value of <0.05 was considered significant.

FIG 3

Fh15 tended to decrease the production of proinflammatory cytokines and chemokines in rhesus monkeys undergoing E. coli infection. Plasma samples were collected from rhesus macaques at different time points, as follows: 0 h (baseline), 30 min, 2 h, 4 h, 6 h, and 8 h. E. coli, comprised animals that only received the i.v. infusion with live E. coli (10¹⁰ CFU/kg body wt.); Fh15-E. coli, comprised animals that received an i.v. infusion containing 12 mg Fh15 followed by the E. coli infection; and Fh15-Fh15-E. coli, comprised animals from the Fh15 group that 3 months later received a second i.v. infusion with 12 mg Fh15 followed by the E. coli infection. Levels of IFN-γ, IL-6, IL-12, TNF-α, MCP-1, and IP10 were measured using Luminex technology. Animals from the Fh15 group do not elicit proinflammatory cytokines (data not shown).

FIG 4

Effect of live E. coli compared with that of Fh15 treatment on the number of innate immune cells in the bloodstream. Graphs represent the number of peripheral blood monocytes, classical monocytes, nonclassical monocytes, intermediate monocytes, dendritic cells, plasmacytoid DCs, natural killer cells, and neutrophils over time in four experimental groups, as follows: E. coli, rhesus macaques (n = 3) that received an i.v. isotonic infusion with live E. coli (10¹⁰ CFU/kg body wt.); Fh15, rhesus macaques (n = 3) that received only i.v. infusion with 12 mg Fh15; Fh15-E. coli, rhesus macaques (n = 3) that received the Fh15 infusion followed by the E. coli infection; and Fh15-Fh15-E. coli, rhesus macaques (n = 3) from the Fh15 that 3 months later received a second infusion with 12 mg Fh15 followed by the E. coli infusion. Whole-blood samples were stained with a cocktail of antibodies for labeling surface markers specific for peripheral monocytes (CD14⁺ and CD16⁺), myeloid and plasmacytoid dendritic cells (CD11c⁺ and CD123⁺), natural killer cells (HLA-DR⁺, CD3⁻, and CD69⁺), and neutrophils (CD66a/c/e⁺). Cells were analyzed by gating using a MacQuant10 instrument. Data were analyzed using FlowJo software. Data represent the average number of cells ± SD of three independent biological samples, with each in duplicate at every time point studied.