Isabelle Allaeys

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA

Significance On activation, blood platelets package components from their cytoplasm into microparticles (MPs), tiny vesicles released by cytoplasmic membrane budding and shedding. Given that MPs can impact other cellular lineages on internalization, we aimed to decipher the mechanisms promoting MP internalization by cellular recipients. We modeled MP internalization by neutrophils and identified a predominant lipid, 12(S)-hydroxyeicosatetranoic acid, as a mediator critical for the promotion of MP internalization. MPs were found inside neutrophils from individuals with rheumatoid arthritis, and their presence in neutrophils in the joints of mice treated with arthritogenic serum is dependent on the expression of enzymes implicated in the generation of 12(S)-hydroxyeicosatetranoic acid. These findings reveal a unique molecular mechanism implicated in MP internalization relevant to inflammatory processes. Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

α2β1 Integrin Regulates Th17 Cell Activity and Its Neutralization Decreases the Severity of Collagen-Induced Arthritis

Th17 cells play a critical role in the pathogenesis of rheumatoid arthritis (RA), but the mechanisms by which these cells regulate the development of RA are not fully understood. We have recently shown that α2β1 integrin, the receptor of type I collagen, is the major collagen-binding integrin expressed by human Th17 cells. In this study, we examined the role of α2β1 integrin in Th17-mediated destructive arthritis in the murine model of collagen-induced arthritis (CIA). We found that α2β1 integrin is expressed on synovial Th17 cells from CIA mice and its neutralization with a specific mAb significantly reduced inflammation and cartilage degradation, and protected the mice from bone erosion. Blockade of α2β1 integrin led to a decrease in the number of Th17 cells in the joints and to a reduction of IL-17 levels in CIA mice. This was associated with an inhibition of receptor activator of NF-κB ligand levels and osteoclast numbers, and reduction of bone loss. We further show that α2β1 integrin is expressed on synovial Th17 cells from RA patients, and that its ligation with collagen costimulated the production of IL-17 by polarized human Th17 cells by enhancing the expression of retinoic acid receptor–related orphan receptor C through ERK and PI3K/AKT. Our findings provide the first evidence, to our knowledge, that α2β1 integrin is an important pathway in Th17 cell activation in the pathogenesis of CIA, suggesting that its blockade can be beneficial for the treatment of RA and other Th17-associated autoimmune diseases.

Picea mariana polyphenolic extract inhibits phlogogenic mediators produced by TNF-α-activated psoriatic keratinocytes: Impact on NF-κB pathway.

ETHNOPHARMACOLOGICAL RELEVANCE Picea mariana ((Miller) Britton, Sterns, and Poggenburg; Pinaceae) bark has been traditionally used by North American natives for treating topical inflammations. It has been also suggested to improve various inflammatory skin disorders like Psoriasis vulgaris. Extracts from this bark storage protein contain polyphenolic compounds which have well-known antiinflammatory activities. Based on the capacity of polyphenolic compounds to modulate functions of normal human keratinocytes, this study was set up to decipher the mechanisms of action of a chemically characterized polyphenolic extract from Picea mariana bark (BS-EAcf) on lesional keratinocytes of skin with psoriasis vulgaris, a disease driven by the immune system in which TNF-α plays a significant role. MATERIALS AND METHODS BS-EAcf corresponds to the ethyl acetate soluble fraction from the hot water extract of Picea mariana bark. BS-EAcf effects were evaluated in normal human (NHK) and psoriatic (PK) keratinocytes stimulated by TNF-α. Cell viability was assessed by lactate deshydrogenase release and propidium iodide (PI) staining. The mechanisms of action of BS-EAcf in keratinocytes were investigated by flow cytometry, ELISAs, RT-PCR and western blot analyses. RESULTS PK exhibited a higher response to TNF-α than NHK regarding the ICAM-1 expression and the production of NO, IL-6, IL-8, fractalkine and PGE2, whereas BS-EAcf significantly inhibited this TNF-α-induced increase at concentrations without causing keratinocyte toxicity. Additionally, this extract significantly inhibited the TNF-α-induced release of elafin and VEGF by PK and NHK. Since TNF-α activation of most of these factors is dependent on the NF-κB pathway, this latter was studied in TNF-α-activated PK. BS-EAcf inhibited the TNF-α-induced phosphorylation and degradation of total IκBα as well as phosphorylation of NF-κB p65. CONCLUSIONS The ethyl acetate fraction from Picea mariana bark extract showed inhibitory effects of cytokines, chemokines, adhesion molecules, nitric oxide and prostaglandins produced by keratinocytes under TNF-α activation through down-regulating the NF-κB pathway. This study demontrated that this extract could be a potential antiinflammatory agent capable of improving psoriatic skin.

NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts

IntroductionMonosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and leucine-rich repeat region containing family of receptor protein 3). MSU is known to activate NLRP3 inflammasomes in professional phagocytes. We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs.MethodsNormal human OBs cultured in vitro were investigated for their capacity for phagocytosis of MSU microcrystals by using confocal microscopy. Subsequent mineralization and matrix metalloproteinase activity were evaluated, whereas regulatory events of phagocytosis were deciphered by using signaling inhibitors, phosphokinase arrays, and small interfering RNAs. Statistics were carried out by using paired or unpaired t tests, and the one-way ANOVA, followed by multiple comparison test.ResultsMost of the OBs internalized MSU in vacuoles. This process depends on signaling via PI3K, protein kinase C (PKC), and spleen tyrosine kinase (Syk), but is independent of Src kinases. Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K. MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3. However, MSU-stimulated autophagy in OBs absolutely requires the phagocytosis process. We find that MSU upregulates NLRP3, which positively controls the formation of MSU-autophagosomes in OBs. MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.ConclusionsMSU microcrystals, found locally encrusted in the bone matrix of chronic gout, activate phagocytosis and NLRP3-dependent autophagy in OBs, but remain intact in permanent autophagosomes while deregulating OB functions.

Osteoblast retraction induced by adherent neutrophils promotes osteoclast bone resorption: implication for altered bone remodeling in chronic gout

Bone destruction in chronic gout is correlated with deposits of monosodium urate (MSU) crystals. Bone with MSU tophi were histopathologically shown to have altered remodeling and cellular distribution. We investigated the impact of neutrophils in bone remodeling associated with MSU and demonstrated that neutrophils, through elastase localized at their surface, induced retraction of confluent osteoblasts (OBs) previously layered on calcified matrix. This OB retraction allowed osteoclasts to resorb cell-free areas of the matrix. This neutrophil effect was concentration dependent and time dependent and required direct contact with OBs. Exposure of OBs to MSU greatly promoted neutrophil adherence to OBs. Neutrophil membrane at the contact zone with OBs showed concentrated fluorescence of dye PKH-67, indicating a cellular contact. Neutrophil–OB interaction increased the survival of neutrophils, reduced their release of lactoferrin in presence of MSU and did not change OB-mediated mineralization. The adhesion of neutrophils to OBs was heterotypic through neutrophil CD29/CD49d and OB-fibronectin peptide CS1. Leukotriene B4 (LTB4) and platelet-activating factor (PAF) were also involved in neutrophil adherence to OBs, as shown by the blocking effect of selective LTB4 and PAF receptor antagonists, and a cytosolic phospholipase A2α (cPLA2α) inhibitor. Blockade of CD49d/CS1 and inhibition of the cPLA2α had subadditive effects, reducing by 60% the adherence of neutrophils to OBs. Taken together, these data showed that neutrophil adhesion to MSU-activated OBs was mediated by the β1 integrin CD29/CD49d-fibronectin peptide CS1 receptors and cPLA2α-derived metabolites and impacts on OB and osteoclast functions. These interactions could be involved in the local bone remodeling process of gout.

Platelets release pathogenic serotonin and return to circulation after immune complex-mediated sequestration

Significance Immune complexes (ICs) form when antibodies encounter their antigens. ICs are present in blood in multiple pathological conditions. Given the abundance of platelets in blood and that they express a receptor for ICs, called Fcγ receptor IIA (FcγRIIA), we examined the impact of ICs in blood in a mouse model. We found that circulating ICs induced systemic shock, characterized by loss of consciousness, by activating platelet FcγRIIA. Shock was mediated by the liberation of serotonin, a molecule better known for its role in the brain, from platelet granules. During shock, platelets were sequestered in the lungs and brain and returned to the blood circulation after their degranulation. Platelets are thus crucial in response to ICs. There is a growing appreciation for the contribution of platelets to immunity; however, our knowledge mostly relies on platelet functions associated with vascular injury and the prevention of bleeding. Circulating immune complexes (ICs) contribute to both chronic and acute inflammation in a multitude of clinical conditions. Herein, we scrutinized platelet responses to systemic ICs in the absence of tissue and endothelial wall injury. Platelet activation by circulating ICs through a mechanism requiring expression of platelet Fcγ receptor IIA resulted in the induction of systemic shock. IC-driven shock was dependent on release of serotonin from platelet-dense granules secondary to platelet outside-in signaling by αIIbβ3 and its ligand fibrinogen. While activated platelets sequestered in the lungs and leaky vasculature of the blood–brain barrier, platelets also sequestered in the absence of shock in mice lacking peripheral serotonin. Unexpectedly, platelets returned to the blood circulation with emptied granules and were thereby ineffective at promoting subsequent systemic shock, although they still underwent sequestration. We propose that in response to circulating ICs, platelets are a crucial mediator of the inflammatory response highly relevant to sepsis, viremia, and anaphylaxis. In addition, platelets recirculate after degranulation and sequestration, demonstrating that in adaptive immunity implicating antibody responses, activated platelets are longer lived than anticipated and may explain platelet count fluctuations in IC-driven diseases.

PKC-δ controls the fMLF-induced overproduction of superoxide by neutrophils

Antimicrobial defense by neutrophils implicates the production of reactive oxygen species. Neutrophil responses can be modulated by agonists such as bacterial peptides and proinflammatory factors that modulate neutrophil activity and survival. We evaluated the production of superoxide anions (O(2)(-)) in response to fMLF by normal human neutrophils after 3 days of preincubation with GM-CSF + IL-4 + TNF-alpha (survival medium). After 3 days of incubation in survival medium, long-lived neutrophils produced up to six times more O(2)(-) relative to control neutrophils in response to fMLF and WKYMVM. This augmented response to fMLF was preferentially linked to formyl peptide receptor (FPR), whereas the response to WKYMVM was dependent on formyl peptide receptor-like 1 (FPRL-1). Real-time RT-PCR revealed a diminution of FPR and FPRL-1 expression in neutrophils incubated in survival medium. fMLF-induced overproduction of O(2)(-) by long-lived neutrophils was independent of intracellular calcium mobilization. The protein tyrosine phosphorylation profile of long-lived neutrophils was modified with respect to control cells. Pharmacological inhibitors of intracellular signals indicated that mechanisms of the excessive fMLF-induced production of O(2)(-) by long-lived neutrophils implicated the protein kinase C (PKC) pathway, preferentially the PKC-delta isoform, whose protein was augmented in these cells. Thus, long-term cytokine exposure modifies molecular pathways and functional characteristics of the neutrophil.

Cooperation between IL-7 Receptor and Integrin α2β1 (CD49b) Drives Th17-Mediated Bone Loss

Th17 cells are critical effectors in inflammation and tissue damage such as bone erosion, but the mechanisms regulating their activation in this process are not fully understood. In this study, we considered the cooperation between cytokine receptors and integrin pathways in Th17-osteoclast function. We found that human Th17 cells coexpress IL-7R and the collagen-binding integrin α2β1 (CD49b), and IL-7 increases their adhesion to collagen via α2β1 integrin. In addition, coengagement of the two receptors in human Th17 cells cooperatively enhanced their IL-17 production and their osteoclastogenic function. The functional cooperation between IL-7R and α2β1 integrin involves activation of the JAK/PI3K/AKT (protein kinase B) and MAPK/ERK pathways. We also showed that IL-7–induced bone loss in vivo is associated with Th17 cell expansion. Moreover, blockade of α2β1 integrin with a neutralizing mAb inhibited IL-7–induced bone loss and osteoclast numbers by reducing Th17 cell numbers in the bone marrow and reducing the production of IL-17 and the receptor activator of NF-κB ligand. Thus, the cooperation between IL-7R and α2β1 integrin can represent an important pathogenic pathway in Th17-osteoclast function associated with inflammatory diseases.

A tagged parathyroid hormone derivative as a carrier of antibody cargoes transported by the G protein coupled PTH1 receptor

Based on the known fact that the parathyroid hormone (PTH) might be extended at its C-terminus with biotechnological protein cargoes, a vector directing the secretion of PTH1-84 C-terminally fused with the antigenic epitope myc (PTH-myc) was exploited. The functional properties and potential of this analog for imaging PTH1R-expressing cells were examined. The PTH-myc construct was recombinantly produced as a conditioned medium (CM) of transfected HEK 293a cells (typical concentrations of 187nM estimated with ELISAs for PTH). PTH-myc CM induced cyclic AMP formations (10min), with a minor loss of potency relative to authentic PTH1-84, and c-Fos expression (1-3h). Treatment of recipient HEK 293a cells transiently expressing PTH1R with PTH-myc CM (supplemented with a fluorescent monoclonal anti-myc tag antibody, either 4A6 or 9E10) allowed the labeling of endosomal structures positive for Rab5 and/or for β-arrestin1 (microscopy, cytofluorometry). Authentic PTH was inactive in this respect, ruling out a non-specific form of endocytosis like pinocytosis. Using a horseradish peroxidase-conjugated secondary antibody, the endocytosis of the PTH-myc-based antibody complex by endogenous PTH1R was evidenced in MG-63 osteoblastoid cells. The secreted construct PTH-myc represents a bona fide agonist that supports the feasibility of transporting cargoes of considerable molecular weight inside cells using arrestin and Rab5-mediated PTH1R endocytosis. PTH-myc is also transported into cells that express PTH1R at a physiological level. Such tagged peptide hormones may be part of a cancer chemotherapy scheme exploiting a modular cytotoxic secondary antibody and the receptor repertoire expressed in a given tumor.

IL-32γ delays spontaneous apoptosis of human neutrophils through MCL-1, regulated primarily by the p38 MAPK pathway.

IL-32γ is a multifunctional cytokine involved in various inflammatory and auto-immune diseases in which neutrophils can affect the evolution of these diseases. To persist at inflammatory sites, neutrophils require inhibition of their rapid and constitutive apoptosis, an inhibitory effect that phlogogenic cytokines support. To date, the effects of IL-32γ on neutrophils remain unknown. We demonstrate that IL-32γ delays, in a dose-dependent manner, the spontaneous apoptosis of human blood neutrophils by activating mainly p38 MAPK through rapid p38 phosphorylation. PI3-K and ERK1/2 MAPK are also involved, but to a lesser extent. Most of cytokines that induce retardation of neutrophil apoptosis activate the expression of MCL-1 at both mRNA and protein levels. IL-32γ added to human blood neutrophils in vitro is associated with sustained levels of MCL-1 protein. This effect in neutrophils corresponds to a decrease of MCL-1 protein degradation without any effect on MCL-1 mRNA levels. The sustained levels of MCL-1 induced by IL-32γ are only abrogated by the p38β MAPK inhibitor SB202190. Additionally, IL-32γ induces a reduction in caspase 3 activity in neutrophils. In conclusion, IL-32γ affects human blood neutrophils in vitro by increasing their survival, suggesting that this cytokine could have profound effects on the deleterious functions of neutrophils in several diseases.