Cynthia Laflamme

Platelets release mitochondria serving as substrate for bactericidal group IIA-secreted phospholipase A2 to promote inflammation

Mitochondrial DNA (mtDNA) is a highly potent inflammatory trigger and is reportedly found outside the cells in blood in various pathologies. Platelets are abundant in blood where they promote hemostasis. Although lacking a nucleus, platelets contain functional mitochondria. On activation, platelets produce extracellular vesicles known as microparticles. We hypothesized that activated platelets could also release their mitochondria. We show that activated platelets release respiratory-competent mitochondria, both within membrane-encapsulated microparticles and as free organelles. Extracellular mitochondria are found in platelet concentrates used for transfusion and are present at higher levels in those that induced acute reactions (febrile nonhemolytic reactions, skin manifestations, and cardiovascular events) in transfused patients. We establish that the mitochondrion is an endogenous substrate of secreted phospholipase A2 IIA (sPLA2-IIA), a phospholipase otherwise specific for bacteria, likely reflecting the ancestral proteobacteria origin of mitochondria. The hydrolysis of the mitochondrial membrane by sPLA2-IIA yields inflammatory mediators (ie, lysophospholipids, fatty acids, and mtDNA) that promote leukocyte activation. Two-photon microscopy in live transfused animals revealed that extracellular mitochondria interact with neutrophils in vivo, triggering neutrophil adhesion to the endothelial wall. Our findings identify extracellular mitochondria, produced by platelets, at the midpoint of a potent mechanism leading to inflammatory responses.

Immunomodulatory impact of the A2A adenosine receptor on the profile of chemokines produced by neutrophils

In LPS‐stimulated human neutrophils, engagement of the adenosine A2A receptor selectively prevented the expression and release of TNF‐α, MIP‐1α/CCL3, MIP‐1β/CCL4, MIP‐2α/CXCl2, and MIP‐3α/CCl20. In mice lacking the A2A receptor, granulocytes that migrated into the air pouch 4 h after LPS injection expressed higher mRNA levels of TNF‐α, MIP‐1α, and MIP‐1β than PMNs from wild‐type mice. In mononuclear cells present in the air pouch 72 h after LPS injection, expression of IL‐1β, TNF‐α, IL‐6, and MCP‐2/CCL6 was higher in A2AR knockout mice. In addition to highlighting neutrophils as an early and pivotal target for mediating adenosine anti‐inflammatory activities, these results identify TNF‐α and the MIP chemokine family as gene products whose expression is pivotally affected by activation of A2AR in LPS‐activated PMNs. Modulation by A2AR in the production of inflammatory signals by PMNs may thus influence the evolution of an inflammatory response by reducing the activation status of inflammatory cells.

Potentiation of neutrophil cyclooxygenase-2 by adenosine: an early anti-inflammatory signal

Neutrophils, which are often the first to migrate at inflamed sites, can generate leukotriene B4 from the 5-lipoxygenase pathway and prostaglandin E2 through the inducible cyclooxygenase-2 pathway. Adenosine, an endogenous autacoid with several anti-inflammatory properties, blocks the synthesis of leukotriene B4 while it potentiates the cyclooxygenase-2 pathway in fMLP-treated neutrophils, following activation of the A2A receptor. Using the murine air pouch model of inflammation, we observed that inflammatory leukocytes from mice lacking the A2A receptor have less cyclooxygenase-2 induction than wild-type animals. In human leukocytes, A2A receptor activation specifically elicited potentiation of cyclooxygenase-2 in neutrophils, but not in monocytes. Signal transduction studies indicated that the cAMP, ERK1/2, PI-3K and p38K intracellular pathways are implicated both in the direct upregulation of cyclooxygenase-2 and in its potentiation. Together, these results indicate that neutrophils are particularly important mediators of adenosines effects. Given the uncontrolled inflammatory phenotype observed in knockout mice and in view of the potent inhibitory actions of prostaglandin E2 on inflammatory cells, an increased cyclooxygenase-2 expression resulting from A2A receptor activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine.

Nucleobindin Co-Localizes and Associates with Cyclooxygenase (COX)-2 in Human Neutrophils

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

Proteinase-activated receptor-2 up-regulation by Fcγ-receptor activation in human neutrophils

We shed new light on the expression and function of the proteinase‐activated receptor (PAR) family, associated with inflammation and hyperalgesia, in human granulocytes. Resting cells expressed constitutive levels of PAR‐2 and PAR‐3 mRNA but not PAR‐1 or PAR‐4. Based on flow cytometry, stimulation with opsonized bacteria (Bop) specifically up‐regulated cell surface expression of PAR‐2 in a concentration‐dependent and time‐dependent manner, independent of transcription or de novo protein synthesis. Primary granules were identified as a source of preformed PAR‐2 that can readily be mobilized at the surface on fusion with the plasma membrane. Cellular response to PAR‐2 activation, measured as changes in intracellular calcium concentration, was enhanced in PAR‐2 up‐regulated cells. Increase of cell‐surface PAR‐2 and of cell responsiveness were dependent specifically on the engagement of immunoglobulin (Ig)‐binding receptors. Together, our results reveal that mobilization of intracellular granules, in response to Ig‐receptor activation, up‐regulates PAR‐2 surface expression and makes neutrophils more responsive to proteinase activity. This enhanced response to PAR‐2 activation indicates that molecular communication between pain and inflammation may be more important than previously believed.—St‐Onge, M., Lagarde, S., Laflamme, C, Rollet‐Labelle, E., Marois, L., Naccache, P. H., Pouliot, M. Proteinase‐activated receptor‐2 up‐regulation by Fcγ‐receptor activation in human neutrophils. FASEB J. 24, 2116–2125 (2010). www.fasebj.org

Oncostatin M decreases interleukin-1 β secretion by human synovial fibroblasts and attenuates an acute inflammatory reaction in vivo

Oncostatin M (OSM) is a pleiotropic cytokine of the IL‐6 family and displays both pro‐inflammatory and anti‐inflammatory activities. We studied the impact of OSM on the gene activation profile of human synovial cells, which play a central role in the progression of inflammatory responses in joints. In synovial cells stimulated with lipopolysaccharide and recombinant human granulocyte‐macrophage colony‐stimulating factor, recombinant human OSM and native OSM secreted by human granulocytes both reduced the gene expression and secretion of IL‐1β and CXCL8, but increased that of IL‐6 and CCL2. This impact on synovial cell activation was not obtained using IL‐6 or leukaemia inhibitory factor. Signal transducer and activator of transcription‐1 appeared to mediate the effects of OSM on stimulated human synovial fibroblasts. In the murine dorsal air pouch model of inflammation, OSM reduced the expression of the pro‐inflammatory cytokines IL‐1β and TNF‐α in lining tissues, and their presence in the cavity. These results as a whole suggest an anti‐inflammatory role for OSM, guiding inflammatory processes towards resolution.

Impact of Anti-Inflammatory Agents on the Gene Expression Profile of Stimulated Human Neutrophils: Unraveling Endogenous Resolution Pathways

Adenosine, prostaglandin E2, or increased intracellular cyclic AMP concentration each elicit potent anti-inflammatory events in human neutrophils by inhibiting functions such as phagocytosis, superoxide production, adhesion and cytokine release. However, the endogenous molecular pathways mediating these actions are poorly understood. In the present study, we examined their impact on the gene expression profile of stimulated neutrophils. Purified blood neutrophils from healthy donors were stimulated with a cocktail of inflammatory agonists in the presence of at least one of the following anti-inflammatory agents: adenosine A2A receptor agonist CGS 21680, prostaglandin E2, cyclic-AMP-elevating compounds forskolin and RO 20-1724. Total RNA was analyzed using gene chips and real-time PCR. Genes encoding transcription factors, enzymes and regulatory proteins, as well as secreted cytokines/chemokines showed differential expression. We identified 15 genes for which the anti-inflammatory agents altered mRNA levels. The agents affected the expression profile in remarkably similar fashion, suggesting a central mechanism limiting cell activation. We have identified a set of genes that may be part of important resolution pathways that interfere with cell activation. Identification of these pathways will improve understanding of the capacity of tissues to terminate inflammatory responses and contribute to the development of therapeutic strategies based on endogenous resolution.

TNF-α expression in neutrophils and its regulation by glycogen synthase kinase-3: A potentiating role for lithium

Glycogen synthase kinase 3 (GSK‐3) is associated with several cellular systems, including immune response. Lithium, a widely used pharmacological treatment for bipolar disorder, is a GSK‐3 inhibitor. GSK‐3α is the predominant isoform in human neutrophils. In this study, we examined the effect of GSK‐3 inhibition on the production of TNF‐α by neutrophils. In the murine air pouch model of inflammation, lithium chloride (LiCl) amplified TNF‐α release. In lipopolysaccharide‐stimulated human neutrophils, GSK‐3 inhibitors mimicked the effect of LiCl, each potentiating TNF‐α release after 4 h, in a concentration‐dependent fashion, by up to a 3‐fold increase (ED50 of 1 mM for lithium). LiCl had no significant effect on cell viability. A positive association was revealed between GSK‐3 inhibition and prolonged activation of the p38/MNK1/eIF4E pathway of mRNA translation. Using lysine and arginine labeled with stable heavy isotopes followed by quantitative mass spectrometry, we determined that GSK‐3 inhibition markedly increases (by more than 3‐fold) de novo TNF‐α protein synthesis. Our findings shed light on a novel mechanism of control of TNF‐α expression in neutrophils with GSK‐3 regulating mRNA translation and raise the possibility that lithium could be having a hitherto unforeseen effect on inflammatory diseases.—Giambelluca, M. S., Bertheau‐Mailhot, G., Laflamme, C., Rollet‐Labelle, E., Servant, M. J., Pouliot, M. TNF‐α expression in neutrophils and its regulation by glycogen synthase kinase‐3: a potentiating role for lithium. FASEB J. 28, 3679–3690 (2014). www.fasebj.org

Age-related decline of the acute local inflammation response: a mitigating role for the adenosine A 2A receptor

Aging is accompanied by an increase in markers of innate immunity. How aging affects neutrophil functions remains of debate. The adenosine A2A receptor (A2AR), essential to the resolution of inflammation, modulates neutrophil functions. We sought to determine whether or not A2AR protects against the effects of aging. We monitored neutrophil influx, viability, and activation as well as cytokine accumulation in wild-type (WT) and A2AR-knockout mice (KO) at three different ages. Several readouts decreased with aging: neutrophil counts in dorsal air pouches (by up to 55%), neutrophil viability (by up to 56%), elastase and total protein in exudates (by up to 80%), and local levels of cytokines (by up to 90%). Each of these parameters was significantly more affected in A2AR-KO mice. CXCL1-3 levels were largely unaffected. The effects of aging were not observed systemically. Preventing neutrophil influx into the air pouch caused a comparable cytokine pattern in young WT mice. Gene expression (mRNA) in leukocytes was affected, with CXCL1 and CCL4 increasing and with TNF and IL-1∝ decreasing. Conclusion: Aging has deleterious effects on the acute inflammatory response and neutrophil-related activities, and defective migration appears as an important factor. A functional A2AR signaling pathway delays some of these.