Alarmins MRP8 and MRP14 Induce Stress Tolerance in Phagocytes under Sterile Inflammatory Conditions

Hyporesponsiveness by phagocytes is a well-known phenomenon in sepsis that is frequently induced by low-dose endotoxin stimulation of Toll-like receptor 4 (TLR4) but can also be found under sterile inflammatory conditions. We now demonstrate that the endogenous alarmins MRP8 and MRP14 induce phagocyte hyporesponsiveness via chromatin modifications in a TLR4-dependent manner that results in enhanced survival to septic shock in mice. During sterile inflammation, polytrauma and burn trauma patients initially present with high serum concentrations of myeloid-related proteins (MRPs). Human neonatal phagocytes are primed for hyporesponsiveness by increased peripartal MRP concentrations, which was confirmed in murine neonatal endotoxinemia in wild-type and MRP14(-/-) mice. Our data therefore indicate that alarmin-triggered phagocyte tolerance represents a regulatory mechanism for the susceptibility of neonates during systemic infections and sterile inflammation

Comparación de la técnica seroneutralización con la inmunoenzimática competitiva en la detección de anticuerpos contra el virus de la estomatitis vesicular (serotipos New Jersey e Indiana) en equinos de Costa Rica

A competitive enzyme-linked immunosorbent assay (c-ELISA) was compared to the serum neutralization test (SNT) for the detection of antibodies against vesicular stomatitis virus, New Jersey (VSV-NJ) and Indiana (VSV-IN) serotypes, using 214 equine serum samples from Costa Rica. In the c-ELISA for VSV-NJ 109 (50.93%) equine sera reacted positive, whereas in the SNT 138 (64.48%) serum samples reacted positive to VSV-NJ. In the c-ELISA for VSV-IN 26 (12.15%) sera reacted positive, whereas in the SNT 36 (16.83%) equine sera reacted positive to VSV-IN. The c-ELISA showed a moderate sensitivity compared to the SNT. The rapidity and ease of the c-ELISA system makes it suitable as a rapid diagnostic screening assay. However, because of the lower sensitivity of the c-ELISA, the SNT is considered the method of choice to detect equine VSV infections in Costa Rica.En este trabajo se comparó la técnica de seroneutralización (SNT) con un ensayo inmunoenzimático competitivo (c-ELISA) para la detección de anticuerpos contra el virus de la estomatitis vesicular, serotipos New Jersey (VSV-NJ) e Indiana (VSV-IN), utilizando 214  sueros equinos de Costa Rica. El c-ELISA detectó 109 (50.93%) sueros positivos; mientras que en SNT se determinó un total de 138 (64,48%) animales positivos a VSV-NJ. Un total de 26 (12.15%) sueros resultó positivos en c-ELISA; mientras que 36 (16,83%) sueros se determinó como positivos en SNT para VSV-IN. La técnica inmunoenzimática mostró una moderada sensibilidad, en comparación con la técnica de seroneutralización. Por su rapidez y fácil ejecución, el ELISA competitivo resulta una técnica importante de tamizaje; sin embargo, debido a la menor sensibilidad del c-ELISA, se considera la SNT como el método de elección para  detectar infecciones equinas con VSV en Costa Rica

Lipid alterations in human blood-derived neutrophils lead to formation of neutrophil extracellular traps

The formation of neutrophil extracellular traps (NETs) as a host innate immune defence mechanism has been shown to be the result of a novel cell death process called NETosis. The objective of this study was to investigate the role of cholesterol in the formation of NETs. To this end, primary human neutrophils were treated with different concentrations of methy-beta-cyclodetxrin (M beta CD) to reduce cholesterol level in the cell. The formation of NETs was studied using immunofluorescence microscopy and Picogreen-quantification of released dsDNA. Neutrophils treated with M beta CD showed a significant release of NETs in a process that is independent of NADPH-oxidase. The effect of M beta CD on the lipid composition of the cells was determined using high performance thin layer chromatography (HPTLC). The identities of lipids separated by HPTLC were confirmed by mass spectrometry. Treatment of neutrophils with M beta CD revealed distinct changes in the lipid composition: The percentage of cholesterol in the cell was significantly reduced; other lipids as sphingomyelin were only slightly affected. Interestingly, neutrophils treated with sphingomyelin-degrading sphingomyelinase also showed significant release of NETs. In conclusion, this study shows that lipid alterations facilitate formation of NETs. (C) 2014 Elsevier GmbH. All rights reserved

Telomere dysfunction promotes small vessel vasculitis via the LL37-NETs-dependent mechanism

Background: Small vessel vasculitis (SVV) is a group of systemic autoimmune diseases that are mediated by neutrophil extracellular traps (NETs) in response to cathelicidin LL37, an aging molecular marker, which could be induced by telomere dysfunction. Therefore, in this study, we evaluated the hypothesis that telomere dysfunction in neutrophils may promote SVV via an LL37-NETs-dependent mechanism. Methods: We contrasted the release of neutrophil NETs from mice with telomere dysfunction, mice with DNA damage and wide-type mice. Neutrophil telomere length, the expression of LL37, and the formation of NETs were measured in SVV patients and healthy controls (HCs). The co-expression of gamma H2AX, LL37, and NETs were detected in SVV patients to evaluate the association of the immune aging of neutrophils and pro-inflammatory conditions. LL37 inhibitor was used to verify its key role in NETs release in SVV patients and DNA damage mice. Results: We found that NETs were over-induced by telomere dysfunction and DNA damage in mice, which may be associated with a marked increase in LL37. For patients with SVV, telomeres in neutrophils were significantly shortened, which was also associated with higher levels of LL37 and NETs. Inhibition of LL37 reduced the NETs released from neutrophils. Conclusions: Taken together, the results of these studies suggest that dysfunction of telomeres may promote SVV through the mechanism of LL37-dependent NETs. Thus, targeting the LL37-NETs may be a novel therapy for SVV.Funding Agencies|National Basic Research Program of ChinaNational Basic Research Program of China [2012CB517603]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China [81470938, 81200546, 81300619]; Zhejiang Science and Technology Department [LQ19H050005, 2012C13G2010133]</p

Novel Role of the Antimicrobial Peptide LL-37 in the Protection of Neutrophil Extracellular Traps against Degradation by Bacterial Nucleases

Neutrophil extracellular traps (NETs) have been described as a fundamental innate immune defence mechanism. They consist of a nuclear DNA backbone associated with different antimicrobial peptides (AMPs) which are able to engulf and kill pathogens. The AMP LL-37, a member of the cathelicidin family, is highly present in NETs. However, the function of LL-37 within NETs is still unknown because it loses its antimicrobial activity when bound to DNA in the NETs. Using immunofluorescence microscopy, we demonstrate that NETs treated with LL-37 are distinctly more resistant to S. aureus nuclease degradation than nontreated NETs. Biochemical assays utilising a random LL-37-fragment library indicated that the blocking effect of LL-37 on nuclease activity is based on the cationic character of the AMP, which facilitates the binding to neutrophil DNA, thus protecting it from degradation by the nuclease. In good correlation to these data, the cationic AMPs human beta defensin-3 and human neutrophil peptide-1 showed similar protection of neutrophil-derived DNA against nuclease degradation. In conclusion, this study demonstrates a novel role of AMPs in host immune defence: beside its direct antimicrobial activity against various pathogens, cationic AMPs can stabilise neutrophil-derived DNA or NETs against bacterial nuclease degradation. (C) 2014 S. Karger AG, Base