A tool to assist rheumatologists to engage their lupus patients: the Purple Butterfly.

Objective: Translating the highly technical medical jargon of SLE into understandable concepts for patients, their families and individuals without expertise in SLE is a serious challenge. To facilitate communication and enable self-management in SLE, we aimed to create an innovative visual tool, the Purple Butterfly. Methods: We selected clinically representative criteria for SLE and transposed them as graphical features in an attractive and meaningful visual. We developed a script in R programming language that automatically transposes clinical data into this visualization. We asked SLE patients from a local cohort about the relevance, usefulness and acceptability of this visual tool in an online pilot survey. Results: The innovative Purple Butterfly features 11 key clinical criteria: age; sex; organ damage; disease activity; comorbidities; use of antimalarials, prednisone, immunosuppressants and biologics; and patient-reported physical and mental health-related quality of life. Each Purple Butterfly provides the health portrait of one SLE patient at one medical visit, and the automatic compilation of the butterflies can illustrate a patient's clinical journey over time. All survey participants agreed that they would like to use the Purple Butterfly to visualize the course of their SLE over time, and 9 of 10 agreed it should be used during their medical consultations. Conclusion: The Purple Butterfly nurtures effective doctor-patient communication by providing concise visual summaries of lupus patients' health conditions. We believe the Purple Butterfly has the potential to empower patients to take charge of their condition, enhance healthcare coordination and raise awareness about SLE.

DUOX2 regulates secreted factors in virus-infected respiratory epithelial cells that contribute to neutrophil attraction and activation.

The first line of defense against respiratory viruses relies on the antiviral and proinflammatory cytokine response initiated in infected respiratory epithelial cells. The cytokine response not only restricts virus replication and spreading, but also orchestrates the subsequent immune response. The epithelial Dual Oxidase 2 (DUOX2) has recently emerged as a regulator of the interferon antiviral response. Here, we investigated the role of DUOX2 in the inflammatory cytokine response using a model of A549 cells deficient in DUOX2 generated using Crispr-Cas9 and infected by Sendai virus. We found that the absence of DUOX2 selectively reduced the induction of a restricted panel of 14 cytokines and chemokines secreted in response to Sendai virus by 20 to 89%. The secreted factors produced by epithelial cells upon virus infection promoted the migration, adhesion, and degranulation of primary human neutrophils, in part through the DUOX2-dependent secretion of TNF and chemokines. In contrast, DUOX2 expression did not impact neutrophil viability or NETosis, thereby highlighting a selective impact of DUOX2 in neutrophil functions. Overall, this study unveils previously unrecognized roles of epithelial DUOX2 in the epithelial-immune cells crosstalk during respiratory virus infection.

IgG-aggregates rapidly upregulate FcgRI expression at the surface of human neutrophils in a FcgRII-dependent fashion: A crucial role for FcgRI in the generation of reactive oxygen species.

Autoimmune complexes are an important feature of several autoimmune diseases such as lupus, as they contribute to tissue damage through the activation of immune cells. Neutrophils, key players in lupus, interact with immune complexes through Fc gamma receptors (FcgR). Incubation of neutrophils with aggregated-IgGs caused degranulation and increased the surface expression of FcgRI within minutes in a concentration-dependent fashion. After 30 minutes, IgG aggregates (1 mg/mL) upregulated FcgRI by 4.95 ± 0.45-fold. FcgRI-positive neutrophils reached 67.24% ± 6.88% on HA-IgGs stimulated neutrophils, from 3.12% ± 1.62% in non-stimulated cells, ranking IgG-aggregates among the most potent known agonists. FcgRIIa, and possibly FcgRIIIa, appeared to mediate this upregulation. Also, FcgRI-dependent signaling proved necessary for reactive oxygen species (ROS) production in response to IgG-aggregates. Finally, combinations of bacterial materials with aggregates dramatically boosted ROS production. This work suggests FcgRI as an essential component in the response of human neutrophils to immune complexes leading to the production of ROS, which may help explain how neutrophils contribute to tissue damage associated with immune complex-associated diseases, such as lupus.

Age-related decline of the acute local inflammation response: a mitigating role for the adenosine A2A 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-1α decreasing.ConclusionAging 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.

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.

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.

Oncostatin M decreases interleukin-1 ß 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-1ß 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-1ß 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.

Proteinase-activated receptor-2 up-regulation by Fcgamma-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.

Impact of anti-inflammatory agents on the gene expression profile of stimulated human neutrophils: unraveling endogenous resolution pathways.

Adenosine, prostaglandin E(2), 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 A(2A) receptor agonist CGS 21680, prostaglandin E(2), 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.

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 Ca(2+)-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 PGE(2) 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 PGE(2) biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE(2). Moreover, neutrophil transfection with hrNuc specifically enhanced PGE(2) biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.

Characterization of prostaglandin E2 generation through the cyclooxygenase (COX)-2 pathway in human neutrophils.

In the present study, we characterized the generation of prostaglandin (PG)E2 in human neutrophils. We found that the Ca2+-dependent type IV cytosolic phospholipase A2 (cPLA2) was pivotally involved in the COX-2-mediated generation of PGE2 in response to a calcium ionophore, as determined by the use of selected PLA2 inhibitors. PGE2 biosynthesis elicited by bacterial-derived peptides or by phagocytic stimuli acting on cell surface receptors also showed to be dependent on cPLA2 activity. We then assessed metabolism of unesterified arachidonic acid (AA), and observed that PGE2 production becomes favored over that of LTB4 with higher AA concentrations. Withdrawal of calcium prevented the generation of PGE2 in response to a calcium ionophore but did not affect the up-regulation of COX-2 or its capacity to convert AA, thus limiting its implication at the level of cPLA2 activation. Of the main eicosanoids produced by neutrophils, only LTB4 was able to up-regulate COX-2 expression. Finally, the only PGE synthase isoform found in neutrophils is microsomal PGE synthase-1; it co-localized with COX-2 and its expression appeared mainly constitutive. These results highlight key differences in regulatory processes of the 5-LO and COX pathways, and enhance our knowledge at several levels in the PGE2 biosynthesis in neutrophils.

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-alpha, MIP-1alpha/CCL3, MIP-1beta/CCL4, MIP-2alpha/CXCL2, and MIP-3alpha/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-alpha, MIP-1alpha, and MIP-1beta than PMNs from wild-type mice. In mononuclear cells present in the air pouch 72 h after LPS injection, expression of IL-1beta, TNF-alpha, 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-alpha 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 B(4) from the 5-lipoxygenase pathway and prostaglandin E(2) through the inducible cyclooxygenase-2 pathway. Adenosine, an endogenous autacoid with several anti-inflammatory properties, blocks the synthesis of leukotriene B(4) while it potentiates the cyclooxygenase-2 pathway in fMLP-treated neutrophils, following activation of the A(2A) receptor. Using the murine air pouch model of inflammation, we observed that inflammatory leukocytes from mice lacking the A(2A) receptor have less cyclooxygenase-2 induction than wild-type animals. In human leukocytes, A(2A) 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 adenosine's effects. Given the uncontrolled inflammatory phenotype observed in knockout mice and in view of the potent inhibitory actions of prostaglandin E(2) on inflammatory cells, an increased cyclooxygenase-2 expression resulting from A(2A) receptor activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine.

Functional characterization of SIX3 homeodomain mutations in holoprosencephaly: interaction with the nuclear receptor NR4A3/NOR1.

Holoprosencephaly (HPE) is a relatively common brain malformation resulting in an incomplete separation of the two cerebral hemispheres. A number of mutations in different genes have been linked to this malformation, including three missense mutations in the homeodomain of the transcription factor SIX3. In this study, we investigated the functional consequences of these SIX3 mutations with respect to the ability of the protein to interact with and stimulate the transcriptional activity of the nuclear receptor NOR1 (NR4A3). Using glutathione S-transferase fusion protein pull-down assays and transient cotransfections of Neuro-2a cells with expression and reporter vectors, we found that one mutation, c.676C>G (p.L226V), does not alter the properties of SIX3 toward NOR1. Another mutation, c.749T>C (p.V250A), results in the production of a highly unstable protein in Neuro-2a cells. The third mutation, c.770G>C (p.R257P), results in a mutant SIX3 protein that no longer interacts with NOR1 in vivo. These observations suggest that different SIX3 mutations in HPE2 may affect different signaling pathways, and that one of these pathways may involve the nuclear receptor NOR1.

The homeotic protein Six3 is a coactivator of the nuclear receptor NOR-1 and a corepressor of the fusion protein EWS/NOR-1 in human extraskeletal myxoid chondrosarcomas.

Nuclear receptors represent a large family of transcription factors involved in development, differentiation, homeostasis, and cancer. In recent years, a growing number of cofactors has been discovered that participate in the regulation of the transcriptional activity of these proteins. We present in this study the identification of a cofactor, the homeotic protein Six3, which differentially regulates the transcriptional activity of the orphan nuclear receptor NOR-1 (NR4A3). NOR-1 is normally involved in the balance between cell proliferation and cell death, and is implicated in oncogenesis as part of the EWS/NOR-1 fusion protein found in human extraskeletal myxoid chondrosarcoma (EMC) tumors. Reverse transcription-PCR analyses indicate that EMC tumors expressing the EWS/NOR-1 mRNA also express mRNAs encoding NOR-1 and Six3. Glutathione S-transferase fusion protein assays show that Six3 binds in vitro the DNA-binding domain of NOR-1 and the EWS domain of EWS/NOR-1 and that the homeodomain of Six3 is required for these interactions. Mammalian two-hybrid experiments, using immortalized human chondrocytes as a model, indicate that Six3 also interacts with NOR-1 and EWS/NOR-1 in vivo. Cotransfection experiments show that Six3 stimulates the transcriptional activity of NOR-1, whereas it represses that of EWS/NOR-1. Considering the highly specific expression pattern of Six3, our finding that it is expressed in EMC suggests that it plays a pivotal role in the development of these tumors. We propose that Six3 maintains a transcriptional balance between the activities of NOR-1 and EWS/NOR-1, the net effect being to deregulate the expression of specific target genes and push the equilibrium toward uncontrolled cell proliferation.