Cytokines induced neutrophil extracellular traps formation: implication for the inflammatory disease condition

Abstract

Neutrophils (PMNs) and cytokines have a critical role to play in host defense and systemic inflammatory response syndrome (SIRS). Neutrophil extracellular traps (NETs) have been shown to extracellularly kill pathogens, and inflammatory potential of NETs has been shown. Microbial killing inside the phagosomes or by NETs is mediated by reactive oxygen and nitrogen species (ROS/RNS). The present study was undertaken to assess circulating NETs contents and frequency of NETs generation by isolated PMNs from SIRS patients. These patients displayed significant augmentation in the circulating myeloperoxidase (MPO) activity and DNA content, while PMA stimulated PMNs from these patients, generated more free radicals and NETs. Plasma obtained from SIRS patients, if added to the PMNs isolated from healthy subjects, enhanced NETs release and free radical formation. Expressions of inflammatory cytokines (IL-1β, TNFα and IL-8) in the PMNs as well as their circulating levels were significantly augmented in SIRS subjects. Treatment of neutrophils from healthy subjects with TNFα, IL-1β, or IL-8 enhanced free radicals generation and NETs formation, which was mediated through the activation of NADPH oxidase and MPO. Pre-incubation of plasma from SIRS with TNFα, IL-1β, or IL-8 antibodies reduced the NETs release. Role of IL-1β, TNFα and IL-8 thus seems to be involved in the enhanced release of NETs in SIRS subjects.

Figure 1. Circulating DNA content, MPO activity and NETs formation in healthy subjects and SIRS patients.

(A) DNA content in the plasma of SIRS patients and control samples (***p<0.001 vs control). (B) MPO activity in plasma of SIRS patients and control subjects (***p<0.001 vs control). (C) MPO activity in PMNs of SIRS patients and control subject. (D) PCR of mitochondrial genes [ATP synthase subunit 6 (atp6), cytochrome oxidase c subunit 1 (co1)], and (E) nuclear genes [Glyceraldehyde-3-phosphate dehydrogenase (gapdh), and β-actin]. (F) Bar diagram representing NETs formation in neutrophils from healthy subjects and SIRS patients following PMA treatment (*p<0.05, ***p<0.001 vs control; ^$$p<0.01, ^$$$p<0.001 vs PMA stimulated cells of healthy volunteer).

Figure 2. NETs formation and free radical generation.

(A) NETs formation following incubation of PMNs from healthy subjects with plasma (20%) from healthy subjects or SIRS patients for 180 min (***p<0.001 vs control, ^$$$p<0.001 vs SIRS). (B) Neutrophil free radical generation in presence of plasma (20%) from healthy subjects and SIRS patients (*p<0.05 vs control).

Figure 3. IL-8, TNFα and IL-1β levels in the plasma and their expression in PMNs.

(A) IL-8, TNFα and IL-1β content as measured by ELISA in the plasma (**p<0.01, ***p<0.001 vs control), and (B) their expression in PMNs by real time RT-PCR (*p<0.05 vs control).