IL-8 Induces Neutrophil Extracellular Trap Formation in Severe Thermal Injury

Abstract

Neutrophil extracellular traps (NETs) have a dual role in the innate immune response to thermal injuries. NETs provide an early line of defence against infection. However, excessive NETosis can mediate the pathogenesis of immunothrombosis, disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) in sepsis. Recent studies suggest that high interleukin-8 (IL-8) levels in intensive care unit (ICU) patients significantly contribute to excessive NET generation. This study aimed to determine whether IL-8 also mediates NET generation in patients with severe thermal injuries. IL-8 levels were measured in serum samples from thermally injured patients with ≥15% of the total body surface area (TBSA) and healthy controls (HC). Ex vivo NET generation was also investigated by treating isolated neutrophils with serum from thermal injured patients or normal serum with and without IL-8 and anti-IL-8 antibodies. IL-8 levels were significantly increased compared to HC on days 3 and 5 (p < 0.05) following thermal injury. IL-8 levels were also significantly increased at day 5 in septic versus non-septic patients (p < 0.001). IL-8 levels were also increased in patients who developed sepsis compared to HC at days 3, 5 and 7 (p < 0.001), day 10 (p < 0.05) and days 12 and 14 (p < 0.01). Serum containing either low, medium or high levels of IL-8 was shown to induce ex vivo NETosis in an IL-8-dependent manner. Furthermore, the inhibition of DNase activity in serum increased the NET-inducing activity of IL-8 in vitro by preventing NET degradation. IL-8 is a major contributor to NET formation in severe thermal injury and is increased in patients who develop sepsis. We confirmed that DNase is an important regulator of NET degradation but also a potential confounder within assays that measure serum-induced ex vivo NETosis.

Keywords: DNase; actin; burns; cell-free DNA (cfDNA); interleukin-8 (IL-8); neutrophil extracellular traps (NETs); sepsis.

Figure 1

IL-8 levels in thermal injury: (A) Shows IL-8 levels in Healthy Control (HC) and burns serum samples from admission to D14, D28, M3, M6, and M12. IL-8 levels were significantly increased on days 3 and 5 compared to HC. (B) A comparison between IL-8 levels in burns patients who developed sepsis or not. IL-8 levels were significantly higher in septic than non-septic burns on D5. (C,D) illustrate the IL-8 level differences for non-septic and septic burns, respectively, compared to HC IL-8 levels. Non-septic burns were not significantly different from HC. In burns patients who developed sepsis, IL-8 levels were significantly increased on days 5–14 compared to HC. p value *** < 0.001, ** < 0.01, * < 0.05. (ns) not significant.

Figure 2

IL-8 induces ex-vivo NETosis. Isolated neutrophils were untreated as a negative control (UT) or stimulated with positive control (PMA), burn patient serum containing a high IL-8 (H IL-8) level (725.49 pg/mL), HC serum, 100 pg/mL recombinant IL-8 or HC serum supplemented with 100 pg/mL IL-8 for 4 h. Induced NETs were labelled with SYTOX Green and anti-citH3 Ab. Composite between cells double labelled with SYTOX and anti-citH3 Ab shows co-localisation. The scale bar represents 0.1 mm. Images are representative of five independent experiments.

Figure 3

NET formation correlates with IL-8 levels in burns serum: (A) Fluorescent microscopy of NETs induced by burns serum containing either high (H), medium (M) or low (L) levels of IL-8, compared to HC serum. Generated NETs were labelled with SYTOX green and an anti-CitH3 Ab. (B) Quantification of NETs in all treatment conditions. (C) CfDNA levels in the supernatants of treated neutrophils. [ANOVA p value] *** < 0.001, ** < 0.01, * < 0.05. (ns) not significant. Data are representative of n = 5.

Figure 4

DNase I inhibition induces more extensive NET formation by serum IL-8: (A) Fluorescent microscopy of neutrophils stimulated with high (H), medium (M) or low (L) IL-8 levels in the absence or presence of 2.5 µM actin. Released chromatin was labelled with an anti-CitH3 Ab. (B) Comparison of NET formation by neutrophils stimulated with H, M, L IL-8, or HC supplemented with 100 pg/mL IL-8 with or without actin. (C) NET formation of H, M and L IL-8 serum. (D) cfDNA levels in burns IL-8 groups with or without actin. [t-test p value]: * < 0.05, ** < 0.01. (ns) not significant. Data are representative of n = 5.

Figure 5

The effect of anti-IL-8 antibodies on NET formation. NET quantification induced by burns serum from high (H), medium (M) or low (L) IL-8 samples, recombinant IL-8, anti-IL-8 Ab isotype, or supplemented HC serum in the presence of 2.5 µM actin and with or without anti-IL-8 Ab. [t-test p value]: * < 0.05. (ns) not significant. Data are representative of n = 5.

Figure 6

The overall IL-8 correlation with cfDNA levels in thermal injuries. Significant overall correlation between IL-8 and cfDNA levels in thermal injuries (n = 96). p value: *** < 0.0001.