Neutrophils Discriminate between Lipopolysaccharides of Different Bacterial Sources and Selectively Release Neutrophil Extracellular Traps

Abstract

The release of neutrophil extracellular traps (NETs), either during “suicidal” or “vital” NETosis, represents an important strategy of neutrophils to combat Gram-negative bacteria. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, is a reported stimulus for NET formation. Although it is widely acknowledged that the structural diversity in LPS structures can elicit heterogeneous immune responses, species- and serotype-specific differences in the capacity of LPS to trigger NET formation have not yet been investigated. In the present study, we compared the NET-inducing potential of LPS derived from Escherichia coli (serotypes O55:B5, O127:B8, O128:B12, O111:B4, and O26:B6), Salmonella enterica (serotype enteritidis), and Pseudomonas aeruginosa (serotype 10), under platelet-free and platelet-rich conditions in vitro, and in whole blood ex vivo. Here, we demonstrate that under serum- and platelet-free conditions, mimicking tissue circumstances, neutrophils discriminate between LPS of different bacterial sources and selectively release NETs only in response to LPS derived from E. coli O128:B12 and P. aeruginosa 10, which both induced “suicidal” NETosis in an autophagy- and reactive oxygen species (ROS)-dependent, but TLR4-independent manner. Intriguingly, in whole blood cultures ex vivo, or in vitro in the presence of platelets, all LPS serotypes induced “vital” NET formation. This platelet-dependent release of NETs occurred rapidly without neutrophil cell death and was independent from ROS formation and autophagy but required platelet TLR4 and CD62P-dependent platelet–neutrophil interactions. Taken together, our data reveal a complex interplay between neutrophils and LPS, which can induce both “suicidal” and “vital” NETosis, depending on the bacterial origin of LPS and the presence or absence of platelets. Our findings suggest that LPS sensing by neutrophils may be a critical determinant for restricting NET release to certain Gram-negative bacteria only, which in turn may be crucial for minimizing unnecessary NET-associated immunopathology.

Cleaved N-terminal histone tails distinguish between NADPH oxidase (NOX)-dependent and NOX-independent pathways of neutrophil extracellular trap formation

“…We have previously shown that elastase has proteolytic activity in NOX-dependent NETs, but not in NOX-independent NETs 10. Why the proteolytic activity of elastase differs between NOX-dependent and NOX-independent NETs is not completely clear, but is likely explained by the differential requirement of NOX-derived ROS during both pathways of NET formation.…”

“…NOX-independent NETs were generated as described previously 10. NETs were either directly visualised and characterised by immunofluorescence microscopy or isolated with 5 U/mL micrococcal nuclease for further characterisation with ELISAs.…”

“…However, the detection of MPO–DNA complexes fails to distinguish between NETs that have been generated through NOX-dependent or NOX-independent pathways, as both contain MPO and DNA 10. Discrimination between NOX-dependent and NOX-independent NETs is important to decipher differences in physiological function, as well as differences in pathophysiological relevance 5.…”

The definition of neutrophil extracellular traps and the concentration of short-chain fatty acids in salmonella-induced inflammation of the intestine against the background of vancomycin and Bacteroides fragilis

“…To introduce the S. enteritidis, S. typhimurium and B. fragilis to rats, we prepared suspensions in the concentration of 1.0 McF standards, which corresponds to the concentration of 3×10 8 CFU/ml. The evaluation of macroscopic manifestations of ileum deficiency was performed on a scale from 0 to 8 in accordance with the criteria of Morris et al in the modification of Jagtap [17]: (0) no visible changes, (1) there are areas of hyperemia, (2) there are lesions having a diameter of 1 mm or less, (3) lesions having a diameter of 2 mm or less (number <5), (4) there is a defect having a diameter of 2 mm or less (number 5-10), (5) a defect having a diameter of 2 mm or less (>10), (6) there is a lesion, having a diameter of more than 2 mm (number <5), (7) there are lesions having a diameter greater than 2 mm (number 5-10); (8) lesions having a diameter greater than 2 mm (>10).…”

“…A recent discovery related to the ability of neutrophils to distinguish lipopolysaccharides from different bacterial agents and selectively release extracellular traps (NETs) is of great interest in studying the mechanisms of interaction of pathogenic microorganisms with immune system cells and their ability to influence the formation of NETs the concentration of SCFA, as a factor affecting the inhibition of inflammatory reactions in the intestine [6,7].…”