A wide variety of microbial and inflammatory factors induce DNA release from neutrophils as neutrophil extracellular traps (NETs). Consensus on the kinetics and mechanism of NET release has been ...hindered by the lack of distinctive methods to specifically quantify NET release in time. Here, we validate and refine a semi-automatic live imaging approach for quantification of NET release. Importantly, our approach is able to correct for neutrophil input and distinguishes NET release from neutrophil death by other means, aspects that are lacking in many NET quantification methods. Real time visualization shows that opsonized S. aureus rapidly induces cell death by toxins, while actual NET formation occurs after 90 minutes, similar to the kinetics of NET release by immune complexes and PMA. Inhibition of SYK, PI3K and mTORC2 attenuates NET release upon challenge with physiological stimuli but not with PMA. In contrast, neutrophils from chronic granulomatous disease patients show decreased NET release only in response to PMA. With this refined method, we conclude that NET release in primary human neutrophils is dependent on the SYK-PI3K-mTORC2 pathway and that PMA stimulation should be regarded as mechanistically distinct from NET formation induced by natural triggers.
Airway-infiltrated neutrophils of children intubated for reasons unrelated to RSV also express LAIR-1 (see Fig E2 in this article's Online Repository at www.jacionline.org)....we find that LAIR-1 is ...expressed on airway-infiltrated neutrophils, but absent on circulating neutrophils, during upper or lower viral respiratory tract infection....RSV loads are already declining when patients are admitted into intensive care.9 Parallels might be drawn with oseltamivir, which needs to be administered within 36 to 48 hours of the onset of symptoms to be effective against influenza....although antibody-mediated ligation of LAIR-1 reduces NET formation by airway-derived neutrophils ex vivo, it is unclear whether LAIR-1 ligation reduces NET formation in vivo or how much NET formation needs to be reduced in vivo to decrease disease severity.
ROS production is an important effector mechanism mediating intracellular killing of microbes by phagocytes. Inappropriate or untimely ROS production can lead to tissue damage, thus tight regulation ...is essential. We recently characterized signal inhibitory receptor on leukocytes‐1 (SIRL‐1) as an inhibitory receptor expressed by human phagocytes. Here, we demonstrate that ligation of SIRL‐1 dampens Fc receptor‐induced ROS production in primary human phagocytes. In accordance, SIRL‐1 engagement on these cells impairs the microbicidal activity of neutrophils, without affecting phagocytosis. The inhibition of ROS production may result from reduced ERK activation, since co‐ligation of Fc receptors and SIRL‐1 on phagocytes inhibited phosphorylation of ERK. Importantly, we demonstrate that microbial and inflammatory stimuli cause rapid downregulation of SIRL‐1 expression on the surface of primary neutrophils and monocytes. In accordance, SIRL‐1 expression levels on neutrophils in bronchoalveolar lavage fluid from patients with neutrophilic airway inflammation are greatly reduced. We propose that SIRL‐1 on phagocytes sets an activation threshold to prevent inappropriate production of oxygen radicals. Upon infection, SIRL‐1 expression is downregulated, allowing microbial killing and clearance of the pathogen.
Signal inhibitory receptor on leukocytes‐1 (SIRL‐1) is an inhibitory receptor with a hitherto unknown ligand, and is expressed on human monocytes and neutrophils. SIRL‐1 inhibits myeloid effector ...functions such as reactive oxygen species (ROS) production. In this study, we identify S100 proteins as SIRL‐1 ligands. S100 proteins are composed of two calcium‐binding domains. Various S100 proteins are damage‐associated molecular patterns (DAMPs) released from damaged cells, after which they initiate inflammation by ligating activating receptors on immune cells. We now show that the inhibitory SIRL‐1 recognizes individual calcium‐binding domains of all tested S100 proteins. Blocking SIRL‐1 on human neutrophils enhanced S100 protein S100A6‐induced ROS production, showing that S100A6 suppresses neutrophil ROS production via SIRL‐1. Taken together, SIRL‐1 is an inhibitory receptor recognizing the S100 protein family of DAMPs. This may help limit tissue damage induced by activated neutrophils.
We identify the S100 protein family of DAMPs as the first known ligands for Signal Inhibitory Receptor on Leukocytes‐1 (SIRL‐1) and show that SIRL‐1 inhibits S100‐induced reactive oxygen species production in human neutrophils.
Abstract
Objectives
Increased release of neutrophil extracellular traps (NETs) is implicated in the activation of plasmacytoid dendritic cells, vascular disease and thrombosis in SLE and APS. ...However, studies comparing NET release between patients with SLE and APS are lacking. Here we evaluated plasma-induced NET release in a large cohort of patients with SLE, SLE + APS and primary APS in relation to clinical and serological parameters.
Methods
Neutrophils from healthy controls were exposed to plasma of heterologous healthy controls (n = 27) or SLE (n = 55), SLE + APS (n = 38) or primary APS (PAPS) (n = 28) patients and NET release was quantified by immunofluorescence. In a subset of SLE patients, NET release was assessed in longitudinal samples before and after a change in treatment.
Results
Plasma-induced NET release was increased in SLE and APS patients, with the highest NET release found in patients with SLE (±APS). Plasma of 60% of SLE, 61% of SLE + APS and 45% of PAPS patients induced NET release. NET release did not correlate with disease activity in SLE or APS. However, increased levels of anti-nuclear and anti-dsDNA autoantibodies were associated with increased NET release in SLE and APS. Only in SLE patients, elevated NET release and an increased number of low-density granulocytes were associated with a high IFN signature.
Conclusion
Increased NET release is associated with autoimmunity and inflammation in SLE and APS. Inhibition of NET release thus could be of potential benefit in a subset of patients with SLE and APS.
Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that ...sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.
During severe respiratory syncytial virus (RSV) bronchiolitis there is a massive influx of activated neutrophils to the lungs. An exaggerated immune response contributes to lung damage and disease ...severity during RSV infection. We have previously shown that normal adult neutrophil function can be modulated by agonists of SIRL-1. Here we aimed to measure the potential of two immune checkpoints: SIRL-1 and LAIR-1, to regulate the function of fresh blood and sputum neutrophils from infants with and without severe RSV bronchiolitis. We show a modest inhibition of the oxidative burst through SIRL-1 and LAIR-1, in control and RSV-infected infants. In addition, SIRL-1 and LAIR-1 inhibited neutrophil extracellular traps (NET) formation by sputum neutrophils of RSV patients. Altogether our data show that inhibitory receptors LAIR-1 and SIRL-1 can be used to regulate neutrophil function.
•Severe respiratory syncytial virus bronchiolitis (RSV) is characterised by a neutrophil influx to the lungs.•Circulating and airway neutrophils from severe RSV bronchiolitis patients show distinct expression of inhibitory receptors.•Neutrophil oxidative burst is regulated by inhibitory receptors in control and RSV infants.
Signal inhibitory receptor on leukocytes‐1 (SIRL‐1) is a negative regulator of myeloid cell function and dampens antimicrobial responses. We here show that different species of the genus ...Staphylococcus secrete SIRL‐1‐engaging factors. By screening a library of single‐gene transposon mutants in Staphylococcus aureus, we identified these factors as phenol‐soluble modulins (PSMs). PSMs are amphipathic α‐helical peptides involved in multiple aspects of staphylococcal virulence and physiology. They are cytotoxic and activate the chemotactic formyl peptide receptor 2 (FPR2) on immune cells. Human cathelicidin LL‐37 is also an amphipathic α‐helical peptide with antimicrobial and chemotactic activities, structurally and functionally similar to α‐type PSMs. We demonstrate that α‐type PSMs from multiple staphylococcal species as well as human cathelicidin LL‐37 activate SIRL‐1, suggesting that SIRL‐1 recognizes α‐helical peptides with an amphipathic arrangement of hydrophobicity, although we were not able to show direct binding to SIRL‐1. Upon rational peptide design, we identified artificial peptides in which the capacity to ligate SIRL‐1 is segregated from cytotoxic and FPR2‐activating properties, allowing specific engagement of SIRL‐1. In conclusion, we propose staphylococcal PSMs and human LL‐37 as a potential new class of natural ligands for SIRL‐1.
Inhibitory and activating immune receptors play a key role in modulating the amplitude and duration of immune responses during infection and in maintaining immune balance in homeostatic conditions. ...The CD200 Receptor (CD200R) gene family in humans encodes one inhibitory receptor, CD200R1, and one putative activating member, CD200R1 Like (CD200R1L). It is demonstrated that CD200R1L is endogenously expressed by human neutrophils and activates cellular functions such as reactive oxygen species (ROS) production via Syk, PI3Kβ, PI3Kδ, and Rac GTPase signaling. Phylogenetic analysis shows that CD200R1L is present in many species among vertebrates, ranging from birds to primates, suggesting that evolutionary conservation of this receptor is critical for protection against co‐evolving pathogens. The duplication event that generated CD200R1L from CD200R occurred several times throughout evolution, supporting convergent evolution of CD200R1L. In our phylogenetic trees, CD200R1L has longer branch lengths than CD200R1 in most species, suggesting that CD200R1L is evolving faster than CD200R1. It is proposed that CD200R1L represents a hitherto uncharacterized activating receptor on human neutrophils.
Graphical
First study to report CD200R1L protein expression in humans; CD200R1L induces production of reactive oxygen species and IL‐8.
The role of the CD200 ligand-CD200 receptor (CD200-CD200R) inhibitory axis is highly important in controlling myeloid cell function. Since the activation of myeloid cells is crucial in ...arteriogenesis, we hypothesized that disruption of the CD200-CD200R axis promotes arteriogenesis in a murine hindlimb ischemia model. Female Cd200-/- and wildtype (C57Bl/6J) mice underwent unilateral femoral artery ligation. Perfusion recovery was monitored over 7 days using Laser-Doppler analysis and was increased in Cd200-/- mice at day 3 and 7 after femoral artery ligation, compared to wildtype. Histology was performed on hindlimb muscles at baseline, day 3 and 7 to assess vessel geometry and number and inflammatory cell influx. Vessel geometry in non-ischemic muscles was larger, and vessel numbers in ischemic muscles were increased in Cd200-/- mice compared to wildtype. Furthermore, T lymphocyte influx was increased in Cd200-/- compared to wildtype. CD200R agonist treatment was performed in male C57Bl/6J mice to validate the role of the CD200-CD200R axis in arteriogenesis. CD200R agonist treatment after unilateral femoral artery ligation resulted in a significant decrease in vessel geometry, perfusion recovery and T lymphocyte influx at day 7 compared to isotype treatment. In this study, we show a causal role for the CD200-CD200R inhibitory axis in arteriogenesis in a murine hindlimb ischemia model. Lack of CD200R signaling is accompanied by increased T lymphocyte recruitment to the collateral vasculature and results in enlargement of preexisting collateral arteries.
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