Fh15 Blocks the Lipopolysaccharide-Induced Cytokine Storm While Modulating Peritoneal Macrophage Migration and CD38 Expression within Spleen Macrophages in a Mouse Model of Septic Shock

Abstract

Sepsis caused by Gram-negative bacteria is the consequence of an unrestrained infection that continuously releases lipopolysaccharide (LPS) into the bloodstream, which triggers an uncontrolled systemic inflammatory response leading to multiorgan failure and death. After scrutinizing the immune modulation exerted by a recombinant Fasciola hepatica fatty acid binding protein termed Fh15, our group demonstrated that addition of Fh15 to murine macrophages 1 h prior to LPS stimulation significantly suppresses the expression of proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL1-β). The present study aimed to demonstrate that Fh15 could exert a similar anti-inflammatory effect in vivo using a mouse model of septic shock. Among the novel findings reported in this article, (i) Fh15 suppressed numerous serum proinflammatory cytokines/chemokines when injected intraperitoneally 1 h after exposure of animals to lethal doses of LPS, (ii) concurrently, Fh15 increased the population of large peritoneal macrophages (LPMs) in the peritoneal cavity (PerC) of LPS-injected animals, and (iii) Fh15 downregulated the expression on spleen macrophages of CD38, a cell surface ectoenzyme with a critical role during inflammation. These findings present the first evidence that the recombinant parasitic antigen Fh15 is an excellent modulator of the PerC cell content and in vivo macrophage activation, endorsing Fh15's potential as a drug candidate against sepsis-related inflammatory response.IMPORTANCE Sepsis is a potentially life-threatening complication of an infection. Sepsis is mostly the consequence of systemic bacterial infections leading to exacerbated activation of immune cells by bacterial products, resulting in enhanced release of inflammatory mediators. Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is a critical factor in the pathogenesis of sepsis, which is sensed by Toll-like receptor 4 (TLR4). The scientific community highly pursues the development of antagonists capable of blocking the cytokine storm by blocking TLR4. We report here that a recombinant molecule of 14.5 kDa belonging to the Fasciola hepatica fatty acid binding protein (Fh15) is capable of significantly suppressing the LPS-induced cytokine storm in a mouse model of septic shock when administered by the intraperitoneal route 1 h after a lethal LPS injection. These results suggest that Fh15 is an excellent candidate for drug development against endotoxemia.

Keywords: CD38; Fasciola hepatica; cytokines; fatty acid binding protein; macrophages; septic shock.

FIG 1

Fh15 suppresses inflammatory cytokines and chemokines in vivo in a murine model of sepsis. Groups of female 6- to 8-week-old BALB/c mice (n = 5) were injected i.p. with 50 μg Fh15 1 h after receiving a lethal i.p. injection with LPS (E. coli O111:B4 [7 mg/kg]). Control mice received PBS, Fh15, or LPS only (i.p.). Mice were sacrificed by cervical dislocation 12 h after LPS exposure, and blood samples were taken by orbital vein or cardiac puncture. A Bioplex mouse cytokine assay was used to measure the concentrations of cytokines (A) and chemokines (B) in serum.

FIG 2

Macroscopic assessment of internal tissues. BALB/c mice injected (i.p.) with Fh15 (50 μg), LPS (E. coli O111:B4 [7 mg/kg]), or Fh15 1 h after LPS injection were sacrificed 12 h after the LPS injection and necropsied to examine the gross appearance of the peritoneal cavity (PerC). Pictures show that the PerC of animals treated with Fh15 exhibits a healthy appearance compared to that of LPS-treated animals.

FIG 3

Fh15 promotes persistence of large peritoneal macrophages in the peritoneal cavity. Peritoneal exudate cells (PECs) were collected from BALB/c mice (6 to 8 weeks old) injected i.p. with 50 μg Fh15 1 h after LPS injection (E. coli O111:B4 [7 mg/kg]), LPS alone, Fh15 alone, or PBS. Cells collected from animals of the same experimental group (n = 5) were pooled and labeled with a cocktail of specific antibodies against CD4⁺, CD8⁺, B220⁺, and NK1.1⁺ to exclude all these cells, including dead cells, by gating. LPMs were identified via their high levels of expression of F4/80 and CD11b. The percentage at the upper right part of the figure represents the amount of LPM relative totals to the cells at the PerC after each treatment. (A) We observed a substantial reduction in the number of LPMs within the LPS-injected group. In contrast, animals treated with Fh15 had a more prevalent LPM population. The data shown are a representative example from an independent experiment. (B) Comparison of the total number of LPMs gated after the different treatments. The data shown represent the average ± SD from three independent experiments. Flow cytometry data were acquired on a Miltenyi MACSQuant Analyzer 10 instrument. Data were analyzed with FlowJo software (FlowJo, LLC).

FIG 4

Fh15 remains in large detectable amounts in the peritoneal cavity of mice. BALB/c mice were i.p. injected with Fh15 (50 μg) conjugated to NIR-VS (Fh15–NIR-VS) (n = 2) or NIR-VS–lysine-conjugated molecule (n = 2). Animals were anesthetized, and the Fh15 distribution was mapped at different time points (30 min and 2 h, 12, and 24 h) after injection using an IVIS Lumina-II (Caliper LifeScience). No significant signal was present in the NIR-VS control mice. In contrast, a strong fluorescence signal from Fh15–NIR-VS was measured in the injected mouse, indicating that Fh15 remained at high concentrations throughout the PerC with a tendency to localize toward the spleen. The image represents the localization of Fh15 24 h after injection.

FIG 5

Fh15 downregulates overexpression of CD38 provoked by LPS insult on the spleen macrophage population. Groups of mice were divided and treated as previously described. After gating, CD38 expression was measured in macrophages (CD11b⁺ F4/80⁺) from spleen. (A) Flow cytometry histogram showing a decrease in CD38 after Fh15 exposure 1 h post-LPS stimulation. (B) Comparison of CD38 expression by mean fluorescent intensity (MFI) values among treatments. (C) Alternative contour plots also showing Fh15’s effect on CD38 expression in LPS-treated mice.

FIG 6

Structure and characteristics of NIR-783-piperazima-vinyl sulfone (NIR-VS). NIR-VS is a synthetic organic compound of 917,183 Da that possesses the property to excite and emit light at 490 and 504 nm, respectively. Fh15 was conjugated to the sulfone groups (S = O) of this compound via free amino groups. This compound was synthesized and kindly donated by the Institute of Biotechnology of the University of Granada, Spain.