Endogenous Tetrapyrroles Influence Leukocyte Responses to Lipopolysaccharide in Human Blood: Pre-Clinical Evidence Demonstrating the Anti-Inflammatory Potential of Biliverdin

Abstract

Sepsis is associated with abnormal host immune function in response to pathogen exposure, including endotoxin (lipopolysaccharide; LPS). Cytokines play crucial roles in the induction and resolution of inflammation in sepsis. Therefore, the primary aim of this study was to investigate the effects of endogenous tetrapyrroles, including biliverdin (BV) and unconjugated bilirubin (UCB) on LPS-induced cytokines in human blood. Biliverdin and UCB are by products of haem catabolism and have strong cytoprotective, antioxidant and anti-inflammatory effects. In the present study, whole human blood supplemented with BV and without was incubated in the presence or absence of LPS for 4 and 8 hours. Thereafter, whole blood was analysed for gene and protein expression of cytokines, including IL-1β, IL-6, TNF, IFN-γ, IL-1Ra and IL-8. Biliverdin (50 μM) significantly decreased the LPS-mediated gene expression of IL-1β, IL-6, IFN-γ, IL-1Ra and IL-8 (P<0.05). Furthermore, BV significantly decreased LPS-induced secretion of IL-1β and IL-8 (P<0.05). Serum samples from human subjects and, wild type and hyperbilirubinaemic Gunn rats were also used to assess the relationship between circulating bilirubin and cytokine expression/production. Significant positive correlations between baseline UCB concentrations in human blood and LPS-mediated gene expression of IL-1β (R=0.929), IFN-γ (R=0.809), IL-1Ra (R=0.786) and IL-8 (R=0.857) were observed in blood samples (all P<0.05). These data were supported by increased baseline IL-1β concentrations in hyperbilirubinaemic Gunn rats (P<0.05). Blood samples were also investigated for complement receptor-5 (C5aR) expression. Stimulation of blood with LPS decreased gene expression of C5aR (P<0.05). Treatment of blood with BV alone and in the presence of LPS tended to decrease C5aR expression (P=0.08). These data indicate that supplemented BV inhibits the ex vivo response of human blood to LPS. Surprisingly, however, baseline UCB was associated with heighted inflammatory response to LPS. This is the first study to explore the effects of BV in a preclinical human model of inflammation and suggests that BV could represent an anti-inflammatory target for the prevention of LPS mediated inflammation in vivo.

Keywords: Cytokine; Inflammation; Lipopolysaccharide; Tetrapyrroles.

Figure 1. Cytokine gene expression in response to LPS and BV

Whole blood was incubated with BV and LPS for 4 h and the mRNA expression was assessed. The relative fold change of each cytokine (A-F) was analysed using 2^{− ΔΔ C}_T method. Data are presented as mean ± S.E. n=7, P<0.05 vs. sample treated with LPS only (0 μM).

Figure 2. Cytokine concentration in response to LPS and BV

Whole blood was incubated with BV and LPS for 8 h and cytokine concentration was measured using a Milliplex human cytokine kit. The relative change in each cytokine (A-F) concentration is presented. Data are presented as mean ± S.E. n=7, P<0.05 vs. sample treated with LPS only (0 μM).

Figure 3. UCB concentration and cytokine gene expression in response to LPS

Whole blood was incubated with BV and LPS for 4 h and mRNA expression was assessed. Figure shows scatter plots and the correlation between baseline UCB concentration and cytokine gene expression (A-F), n=7.

Figure 4. UCB concentration and cytokine concentration in response to LPS

Whole blood was incubated with BV and LPS for 8 h and plasma cytokine concentration was measured using a Milliplex human cytokine kit. Figure shows scatter plots and the correlation between baseline UCB concentration and plasma cytokine concentrations (A-F), n=7.

Figure 5. IL-1β concentration in blood samples of wild type control and Gunn rats

(A) Graph showing the body weight of Wistar (n=10) and Gunn rats (n=17). Data are presented as mean ± S.E; P<0.05 vs control (non-jaundiced Wister rats). Box plot showing the serum UCB concentration (B) and IL-1β concentration in Wistar and Gunn rats. (C) Data are presented as median (25-75% interquartile range); n=10 for Wister and n =17 for Gunn rats and P<0.05 vs. control (non-jaundiced Wister rats). (D) Scatter plot and the correlation between baseline UCB concentration and IL-1β concentration; n=10 for Wister and n =17 for Gunn rats.

Figure 6. IL-8 concentration in response to LPS and BV

IL-8 gene and protein concentration was analysed using qPCR and high sensitivity ELISA kit, respectively in blood samples incubated with BV and LPS for 8 h. IL-8 gene expression (A) and plasma release (B) in response to BV + LPS. Data are presented as mean ± S.E. n=7 and P<0.05 vs. sample treated with LPS only (0 μM). Scatter plot showing the correlation between baseline UCB concentration and IL-8 gene (C) and protein expression (D) in response to LPS, n=7.

Figure 7. Possible mechanism of BV and UCB-triggered immune-modulatory effects

Haem is catabolised into BV, iron (Fe⁺⁺) and carbon monoxide (CO) via the action of haem oxygenase (HO). Biliverdin is rapidly reduced to UCB in the presence of BVR. Pro-inflammatory mediators and endotoxin activate NF-κB p60/p65 dimer and promote its translocation to the nucleus, where it induces the transcription and translation of pro-inflammatory genes. Biliverdin inhibits the expression of pro-inflammatory mediators via inhibition of NF-κB activation. However UCB, similar to dioxins, may promote translocation of AhR from the cytoplasm and binding to xenobiotics/dioxin responsive elements, which results in activation of AhR. Activated form of AhR then leads to increase expression of cytokines (TNF and IL-1β).