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Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. As pathogen-derived neuraminidase (NEU) stimulate neutrophils, we investigated whether host NEU can be targeted to regulate neutrophil dysregulation observed in severe infections.The effects of NEU inhibitors in lipopolysaccharide (LPS)-stimulated neutrophils from healthy donors or COVID-19 patients were determined by evaluating the shedding of surface sialic acids, cell activation, and reactive oxygen species (ROS) production. Re-analysis of single-cell RNA sequencing of respiratory tract samples from COVID-19 patients was also carried out. The effects of Oseltamivir on sepsis and betacoronavirus-induced acute lung injury were evaluated in murine models.Oseltamivir and Zanamivir constrain host NEU activity, surface sialic acid release, cell activation, and ROS production by LPS-activated human neutrophils. Mechanistically, LPS increased the interaction of NEU1 with the matrix metalloproteinase (MMP)-9. Inhibition of MMP-9 prevents LPS-induced NEU activity and neutrophil response. In vivo, treatment with Oseltamivir fine-tunes neutrophil migration and improves infection control and host survival in peritonitis and pneumonia sepsis. NEU1 is also highly expressed in neutrophils from COVID-19 patients and treatment of whole blood samples from these patients with Oseltamivir or Zanamivir reduces neutrophil overactivation. Oseltamivir treatment of intranasally infected mice with the mouse hepatitis coronavirus 3 (MHV-3) decreased lung neutrophil infiltration, viral load, and tissue damage.These findings suggest that NEU1-MMP-9 interplay induces neutrophil overactivation. In vivo, it was shown that NEU may serve as a host-directed target to dampen neutrophil dysfunction during severe infections.

Superoxide production by the phagocyte reduced NAD phosphate (NADPH) oxidase is essential for innate immunity as shown in chronic granulomatous disease (CGD), an immunodeficiency disease resulting from mutations in 1 of its genes. The NADPH oxidase is composed of 2 membrane proteins (gp91phox/NOX2 and p22phox) and 4 cytosolic proteins (p47phox, p67phox, p40phox, and Rac1/2). The phosphorylation of p47phox is required for NADPH oxidase activation in cells. As p47phox and p67phox can form a tight complex in cells, we hypothesized that p67phox could regulate p47phox phosphorylation. To investigate this hypothesis, we used phospho-specific antibodies against 5 major p47phox-phosphorylated sites (Ser304, Ser315, Ser320, Ser328, and Ser345) and neutrophils from healthy donors and from p67phox−/− CGD patients. Results showed that formyl-methionyl-leucyl-phenylalanine and phorbol myristate acetate induced a time- and a concentration-dependent phosphorylation of p47phox on Ser304, Ser315, Ser320, and Ser328 in healthy human neutrophils. Interestingly, in neutrophils and Epstein-Barr virus-transformed B lymphocytes from p67phox−/− CGD patients, phosphorylation of p47phox on serine residues was dramatically reduced. In COSphox cells, the presence of p67phox led to increased phosphorylation of p47phox. In vitro studies showed that recombinant p47phox was phosphorylated on Ser304, Ser315, Ser320, and Ser328 by different PKC isoforms and the addition of recombinant p67phox alone or in combination with p40phox potentiated this process. Thus, p67phox and p40phox are required for optimal p47phox phosphorylation on Ser304, Ser315, Ser320, and Ser328 in intact cells. Therefore, p67phox and p40phox are novel regulators of p47phox-phosphorylation.

Monocytes play a key role in innate immunity by eliminating pathogens, releasing high levels of cytokines, and differentiating into several cell types, including macrophages and dendritic cells. Similar to other phagocytes, monocytes produce superoxide anions through the NADPH oxidase complex, which is composed of two membrane proteins (p22phox and gp91phox/NOX2) and four cytosolic proteins (p47phox, p67phox, p40phox and Rac1). The pathways involved in NADPH oxidase activation in monocytes are less known than those in neutrophils. Here, we show that p22phox is associated with Rho-associated coiled-coil kinase 2 (ROCK2) in human monocytes but not neutrophils. This interaction occurs between the cytosolic region of p22phox (amino acids 132 to 195) and the coiled-coil region of ROCK2 (amino acids 400 to 967). Interestingly, ROCK2 does not phosphorylate p22phox, p40phox, p67phox, or gp91phox in vitro but phosphorylates p47phox on Ser304, Ser315, Ser320 and Ser328. Furthermore, KD025, a selective inhibitor of ROCK2, inhibited reactive oxygen species (ROS) production and p47phox phosphorylation in monocytes. Specific inhibition of ROCK2 expression in THP1-monocytic cell line by siRNA inhibited ROS production. These data show that ROCK2 interacts with p22phox and phosphorylates p47phox, and suggest that p22phox could be a shuttle for ROCK2 to allow p47phox phosphorylation and NADPH oxidase activation in human monocytes.

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils.In vivo, treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.At a GlanceIn a severe systemic inflammatory response, such as sepsis and COVID-19, neutrophils play a central role in organ damage. Thus, finding new ways to inhibit the exacerbated response of these cells is greatly needed. Here, we demonstrate thatin vitrotreatment of whole blood with the viral neuraminidase inhibitors Oseltamivir or Zanamivir, inhibits the activity of human neuraminidases as well as the exacerbated response of neutrophils. In experimental models of severe sepsis, oseltamivir decreased neutrophil activation and increased the survival rate of mice. Moreover, Oseltamivir or Zanamivirex vivotreatment of whole blood cells from severe COVID-19 patients rewire neutrophil function.

Neutrophils are key players in the hyperinflammatory response upon SARS-CoV-2 infection. We have previously described that cytosolic proliferating cell nuclear antigen (PCNA) controls neutrophil survival and NADPH oxidase-dependent ROS production. We here show that both PCNA and S100A8 expression and interaction were elevated in neutrophils from patients with COVID-19 compared to healthy donors and this was correlated with disease severity. Increased PCNA expression was accompanied by a decreased apoptosis and increased NADPH-oxidase activity in neutrophils from COVID-19 patients compared to healthy donors. These effects, as well as the interaction between PCNA and S100A8, were potently counteracted by T2 amino alcohol (T2AA), a PCNA inhibitor, demonstrating that the PCNA scaffold orchestrated neutrophil activation. Notably, the interaction between PCNA-S100A8 was more intense in the CD16high-CD62Llowactivated neutrophil subset. We propose that PCNA-S100A8 complex acts as potential driver for neutrophil dysregulation in COVID-19 and show for the first time that the PCNA scaffold is a decisive component of both neutrophil activation and heterogeneity.

Neutrophils play a pivotal role in innate immunity and in the inflammatory response. Neutrophils are very motile cells that are rapidly recruited to the inflammatory site as the body first line of defense. Their bactericidal activity is due to the release into the phagocytic vacuole, called phagosome, of several toxic molecules directed against microbes. Neutrophil stimulation induces release of this arsenal into the phagosome and induces the assembly at the membrane of subunits of the NAPDH oxidase, the enzyme responsible for the production of superoxide anion that gives rise to other reactive oxygen species (ROS), a process called respiratory burst. Altogether, they are responsible for the bactericidal activity of the neutrophils. Excessive activation of neutrophils can lead to extensive release of these toxic agents, inducing tissue injury and the inflammatory reaction. Envenomation, caused by different animal species (bees, wasps, scorpions, snakes etc.), is well known to induce a local and acute inflammatory reaction, characterized by recruitment and activation of leukocytes and the release of several inflammatory mediators, including prostaglandins and cytokines. Venoms contain several molecules such as enzymes (phospholipase A2, L-amino acid oxidase and proteases, among others) and peptides (disintegrins, mastoporan, parabutoporin etc.). These molecules are able to stimulate or inhibit ROS production by neutrophils. The present review article gives a general overview of the main neutrophil functions focusing on ROS production and summarizes how venoms and venom molecules can affect this function.

Immunoglobulin light chains are called free when they are not linked with heavy chains to form a whole immunoglobulin. Quantification of free light chains is a part of the French national authority for health guidelines for diagnostic and follow-up of light chain, oligo or non-secretory myeloma and AL amyloidosis. Most recently, the World health organisation had included free light chains quantification in prognostic criteria for monoclonal gammopathy of undetermined significance. However the literature bring to light some other potential indications of this analysis in the exploration of monoclonal gammopathy, also in lymphoid malignancies and some autoimmune diseases such as diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis and Sjögren syndrome.

COVID-19 can cause acute respiratory distress syndrome (ARDS), leading to death in a significant number of individuals. Evidence of a strong role of the innate immune system is accumulating, but the precise cells and mechanism involved remain unclear. In this study, we investigated the links between circulating innate phagocyte phenotype and functions and severity in COVID-19 patients. Eighty-four consecutive patients were included, 44 of which were in intensive care units (ICU). We performed an in-depth phenotyping of neutrophil and monocyte subpopulations and measured soluble activation markers in plasma. Additionally, myeloid cell functions (phagocytosis, oxidative burst, and NETosis) were evaluated on fresh cells from patients. Resulting parameters were linked to disease severity and prognosis. Both ICU and non-ICU patients had circulating neutrophils and monocytes with an activated phenotype, as well as elevated concentrations of soluble activation markers (calprotectin, myeloperoxidase, neutrophil extracellular traps, MMP9, sCD14) in their plasma. ICU patients were characterized by increased CD10low CD13low immature neutrophils, LOX-1+ and CCR5+ immunosuppressive neutrophils, and HLA-DRlow CD14low downregulated monocytes. Markers of immature and immunosuppressive neutrophils were strongly associated with severity and poor outcome. Moreover, neutrophils and monocytes of ICU patients had impaired antimicrobial functions, which correlated with organ dysfunction, severe infections, and mortality. Our study reveals a marked dysregulation of innate immunity in COVID-19 patients, which was correlated with severity and prognosis. Together, our results strongly argue in favor of a pivotal role of innate immunity in COVID-19 severe infections and pleads for targeted therapeutic options.One Sentence SummaryOur study reveals a marked dysregulation of innate immunity in COVID-19 patients, which correlates with severity and prognosis.

International audience; Immune system dysfunction is paramount in coronavirus disease 2019 (COVID-19) severity and fatality rate. Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in mucosal immunity and protection against viral infections. Here, we studied the immune cell landscape, with emphasis on MAIT cells, in cohorts totaling 208 patients with various stages of disease. MAIT cell frequency is strongly reduced in blood. They display a strong activated and cytotoxic phenotype that is more pronounced in lungs. Blood MAIT cell alterations positively correlate with the activation of other innate cells, proinflammatory cytokines, notably interleukin (IL)-18, and with the severity and mortality of severe acute respiratory syndrome coronavirus 2 infection. We also identified a monocyte/macrophage interferon (IFN)-α-IL-18 cytokine shift and the ability of infected macrophages to induce the cytotoxicity of MAIT cells in an MR1-dependent manner. Together, our results suggest that altered MAIT cell functions due to IFN-α-IL-18 imbalance contribute to disease severity, and their therapeutic manipulation may prevent deleterious inflammation in COVID-19 aggravation.

Neutrophils play a pivotal role in innate immunity and in the inflammatory reactions. Upon activation, neutrophils release several toxic molecules directed against microbial pathogens into the phagosome. These molecules include reactive oxygen species (ROS), myeloperoxidase, glucosidases, proteases, and antibacterial peptides. In resting cells these proteins and the enzyme responsible for ROS production (NOX2) are stored inside or at the membranes of different granules called azurophil or primary, specific or secondary, gelatinase or tertiary, and the secretory vesicles. Each granule has a specific density, content, and markers. Myeloperoxidase (MPO) is the azurophil granule marker, and the neutrophil-gelatinase-associated lipocalin (NGAL) is the specific granule marker. After cell activation by different stimuli, granule contents are released into the phagosome or in the extracellular space through a process called degranulation. Also during this process, membrane granules fuse with the phagosome and plasma membrane allowing expression of new markers at the cell surface. The degranulation can be assessed by measuring either the release of different proteins by neutrophils or the expression of granule markers at the plasma membrane. In this chapter, we describe the techniques used to measure degranulation of azurophil and specific neutrophil granules using different approaches such as measurement of MPO enzymatic activity and detection of MPO and NGAL proteins by SDS-PAGE and Western blot.

Neutrophils play a pivotal role in innate immunity and in the inflammatory reactions. Upon activation, neutrophils release several toxic molecules directed against microbial pathogens into the phagosome. These molecules include reactive oxygen species (ROS), myeloperoxidase, glucosidases, proteases, and antibacterial peptides. In resting cells these proteins and the enzyme responsible for ROS production (NOX2) are stored inside or at the membranes of different granules called azurophil or primary, specific or secondary, gelatinase or tertiary, and the secretory vesicles. Each granule has a specific density, content, and markers. Myeloperoxidase (MPO) is the azurophil granule marker, and the neutrophil-gelatinase-associated lipocalin (NGAL) is the specific granule marker. After cell activation by different stimuli, granule contents are released into the phagosome or in the extracellular space through a process called degranulation. Also during this process, membrane granules fuse with the phagosome and plasma membrane allowing expression of new markers at the cell surface. The degranulation can be assessed by measuring either the release of different proteins by neutrophils or the expression of granule markers at the plasma membrane. In this chapter, we describe the techniques used to measure degranulation of azurophil and specific neutrophil granules using different approaches such as measurement of MPO enzymatic activity and detection of MPO and NGAL proteins by SDS-PAGE and Western blot.

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22PHOX, and the cytosolic proteins, NOXO1, NOXA1, and Rac1. Here, we investigated whether NOX1 activation and ROS production induced by key inflammatory cytokines in IBD causally affects LCN-2 production in colonic epithelial cells. We found that the combination of TNFα and IL-17 induced a dramatic upregulation of NOXO1 expression that was dependent on the activation of p38MAPK and JNK1/2, and resulted into an increase of NOX1 activity and ROS production. NOX1-derived ROS drive the expression of LCN-2 by controlling the expression of IκBζ, a master inducer of LCN-2. Furthermore, LCN-2 production and colon damage were decreased in NOX1-deficient mice during TNBS-induced colitis. Finally, analyses of biopsies from patients with Crohn's disease showed increased JNK1/2 activation, and NOXO1 and LCN-2 expression. Therefore, NOX1 might play a key role in mucosal immunity and inflammation by controlling LCN-2 expression.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Introduction L’atteinte pulmonaire de l’infection a SARS-CoV-2, responsable du COVID-19, est grave dans 15–20% des cas, avec une pneumopathie bilaterale presentant les caracteristiques histologiques d’un dommage alveolaire diffus. Les fibrocytes circulants, precurseurs des fibroblastes impliques dans la reparation tissulaire, sont associes a un mauvais pronostic dans les maladies fibrosantes pulmonaires et le syndrome de detresse respiratoire aigue. Notre objectif etait de quantifier les fibrocytes circulants chez les patients atteints de COVID et de determiner leur valeur pronostique. Methodes Les fibrocytes circulants (CD45 + /CD15 -/CD34 + /Collagene-1 +) ont ete quantifies en cytometrie de flux chez 57 patients (42 hommes, âge moyen 60 ans [3–87]) hospitalises pour pneumopathie bilaterale hypoxemiante et 15 sujets sains apparies pour l’âge et le sexe. L’infection a SARS-CoV-2 etait confirmee par une PCR naso-pharyngee. L’etendue scannographique des lesions a ete quantifiee. Resultats Le taux median de fibrocytes circulants etait significativement augmente chez les patients par rapport aux temoins (2,49% vs 1,82%, p 75%: 0,57%). Le % de fibrocytes etait diminue lorsqu’une corticotherapie systemique etait deja en place au moment du prelevement (2,28% vs 2,82%, p = 0,04). Un taux de fibrocytes significativement plus bas (1,25%) etait observe chez les patients decedes (6/57) par rapport aux patients rentres a domicile a J28 (2,52%, p = 0,03). Concernant l’evolution scannographique a 3 mois, une regression complete des lesions (13/32) etait associee a un % initial de fibrocytes significativement plus eleve par rapport aux patients presentant une regression partielle ou une stabilite (2,95% vs 2,18%, p = 0,03). Conclusion Le taux de fibrocytes circulants etait significativement augmente chez les patients hospitalises pour une pneumopathie hypoxemiante COVID-19 par rapport a des sujets sains. Les patients ayant l’atteinte la plus severe avaient des % de fibrocytes plus bas, alors qu’un % de fibrocytes plus eleve etait associe a une regression complete des lesions scannographique a 3 mois.

Polymorphonuclear neutrophils are key players in host defense against pathogens through the robust production of superoxide anion by the NADPH oxidase and the release of antibacterial proteins from granules. However, inappropriate release of these agents in the extracellular environment induces severe tissue injury, thereby contributing to the physiopathology of acute and chronic inflammatory disorders. Many studies have been carried out to identify molecules capable of inhibiting phagocyte functions, in particular superoxide anion production, for therapeutic purposes. In the present study, we show that thymoquinone (TQ), the major component of the volatile oil from Nigella sativa (black cumin) seeds strongly inhibits fMLF-induced superoxide production and granules exocytosis in neutrophils. The inhibition of superoxide anion was not due to a scavenger effect, as TQ did not inhibit superoxide anion produced by the xanthine/xanthine oxidase system. Interestingly, TQ impaired the phosphorylation on Ser-304 and Ser-328 of p47(PHOX), a cytosolic subunit of the NADPH oxidase. TQ also attenuated specific and azurophilic granule exocytosis in fMLF-stimulated neutrophils as evidenced by decreased cell surface expression of gp91(PHOX) and CD11b, and release of myeloperoxidase. Furthermore, both the PKC and MAPK pathways, which are involved in p47(PHOX) phosphorylation and granules exocytosis, respectively, were inhibited by TQ in fMLF-stimulated neutrophils. Finally, in a model of pleurisy induced by λ-carrageenan in rats, TQ reduced neutrophil accumulation in the pleural space, showing that it not only inhibits PMN functions in vitro, but also exhibits anti-inflammatory properties in vivo. Thus, TQ possesses promising anti-inflammatory therapeutic potential.

OBJECTIVES Alveolar macrophage polarization and role on alveolar repair during human acute respiratory distress syndrome remain unclear. This study aimed to determine during human acute respiratory distress syndrome: the alveolar macrophage polarization, the effect of alveolar environment on macrophage polarization, and the role of polarized macrophages on epithelial repair. DESIGN Experimental ex vivo and in vitro investigations. SETTING Four ICUs in three teaching hospitals. PATIENTS Thirty-three patients with early moderate-to-severe acute respiratory distress syndrome were enrolled for assessment of the polarization of alveolar macrophages. INTERVENTIONS Polarization of acute respiratory distress syndrome macrophages was studied by flow cytometry and quantitative polymerase chain reaction. Modulation of macrophage polarization was studied in vitro using phenotypic and functional readouts. Macrophage effect on repair was studied using alveolar epithelial cells in wound healing models. MEASUREMENTS AND MAIN RESULTS Ex vivo, alveolar macrophages from early acute respiratory distress syndrome patients exhibited anti-inflammatory characteristics with high CD163 expression and interleukin-10 production. Accordingly, early acute respiratory distress syndrome-bronchoalveolar lavage fluid drives an acute respiratory distress syndrome-specific anti-inflammatory macrophage polarization in vitro, close to that induced by recombinant interleukin-10. Culture supernatants from macrophages polarized in vitro with acute respiratory distress syndrome-bronchoalveolar lavage fluid or interleukin-10 and ex vivo acute respiratory distress syndrome alveolar macrophages specifically promoted lung epithelial repair. Inhibition of the hepatocyte growth factor pathway in epithelial cells and hepatocyte growth factor production in macrophages both reversed this effect. Finally, hepatocyte growth factor and soluble form of CD163 concentrations expressed relatively to macrophage count were higher in bronchoalveolar lavage fluid from acute respiratory distress syndrome survivors. CONCLUSIONS Early acute respiratory distress syndrome alveolar environment drives an anti-inflammatory macrophage polarization favoring epithelial repair through activation of the hepatocyte growth factor pathway. These results suggest that macrophage polarization may be an important step for epithelial repair and acute respiratory distress syndrome recovery.

Inflammatory bowel disease (IBD) is characterized by severe and recurrent inflammation of the gastrointestinal tract, associated with altered patterns of cytokine synthesis, excessive reactive oxygen species (ROS) production, and high levels of the innate immune protein, lipocalin-2 (LCN-2), in the mucosa. The major source of ROS in intestinal epithelial cells is the NADPH oxidase NOX1, which consists of the transmembrane proteins, NOX1 and p22

Background Henoch-Schonlein purpura, more recently renamed immunoglobulin A vasculitis (IgAV), is a systemic vasculitis characterized by IgA deposits. The current markers used to assess IgAV inaccurately evaluate the risk of nephritis occurrence and its long-term outcomes. The current study assessed biomarkers of nephritis outcomes. Methods This French multicentre prospective study enrolled 85 adult patients at the time of disease onset. Patients were assessed for clinical and biological parameters and re-examined after 1 year. Immunoglobulins, cytokines, IgA glycosylation, IgA complexes and neutrophil gelatinase-associated lipocalin (NGAL) concentrations were assessed in blood and urine. Results We identified 60 patients with IgAV-related nephritis (IgAV-N) and 25 patients without nephritis (IgAV-woN). At the time of inclusion (Day 1), the serum levels of galactose-deficient IgA1 (Gd-IgA1) and urinary concentrations of IgA, IgG, IgM, NGAL, interleukin (IL)-1β, IL-6, IL-8, IL-10, IgA-IgG and IgA-sCD89 complexes were higher in the IgAV-N patients than in the IgAV-woN patients (P

Objective Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine–based activation motif (ITAMi) configuration through SH2 domain–containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling. Methods The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody–induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase. Results Treatment with mIgA impaired cell activation in an FcαRI–FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration. Conclusion These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

Neutrophils play a key role in host defense against invading pathogens by releasing toxic agents, such as ROS and antimicrobial peptides. Human neutrophils express several TLRs that recognize a variety of microbial motifs. The interaction between TLR and their agonists is believed to help neutrophils to recognize and to kill pathogens efficiently by increasing their activation, a process called priming. However, excessive activation can induce tissue injury and thereby, contribute to inflammatory disorders. Agonists that activate TLR7 and TLR8 induce priming of neutrophil ROS production; however, which receptor is involved in this process has not been elucidated. In this study, we show that the selective TLR8 agonist, CL075 (3M002), induced a dramatic increase of fMLF-stimulated NOX2 activation, whereas the selective TLR7 agonist, loxoribine, failed to induce any priming effect. Interestingly, CL075, but not loxoribine, induced the phosphorylation of the NOX2 cytosolic component p47phox on several serines and the phosphorylation of p38MAPK and ERK1/2. The inhibitor of p38MAPK completely blocked CL075-induced phosphorylation of p47phox Ser345. Moreover, CL075, but not loxoribine, induced the activation of the proline isomerase Pin1, and juglone, a Pin1 inhibitor, prevented CL075-mediated priming of fMLF-induced superoxide production. These results indicate that TLR8, but not TLR7, is involved in priming of human neutrophil ROS production by inducing the phosphorylation of p47phox and p38MAPK and that Pin1 is also involved in this process.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by failure of superoxide production in phagocytic cells. The disease is characterised by recurrent infections and inflammatory events, frequently affecting the lungs. Improvement of life expectancy now allows most patients to reach adulthood. We aimed to describe the pattern of pulmonary manifestations occurring during adulthood in CGD patients.This was a retrospective study of the French national cohort of adult patients (≥16 years old) with CGD.Medical data were obtained for 67 adult patients. Pulmonary manifestations affected two-thirds of adult patients. Their incidence was significantly higher than in childhood (mean annual rate 0.22 versus 0.07, p=0.01). Infectious risk persisted despite anti-infectious prophylaxis. Invasive fungal infections were frequent (0.11 per year per patient) and asymptomatic in 37% of the cases. They often required lung biopsy for diagnosis (10 out of 30). Noninfectious respiratory events concerned 28% of adult patients, frequently associated with a concomitant fungal infection (40%). They were more frequent in patients with the X-linked form of CGD. Immune-modulator therapies were required in most cases (70%).Respiratory manifestations are major complications of CGD in adulthood. Noninfectious pulmonary manifestations are as deleterious as infectious pneumonia. A specific respiratory monitoring is necessary.

Reactive oxygen species (ROS) are produced by numerous biological systems and by several phagocytes such as neutrophils and macrophages. ROS include mostly superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radical, which are involved in a variety of biological processes such as immunity, inflammation, apoptosis and cell signaling. Thus, there is a need for a sensitive and reliable method to measure ROS. The luminol-amplified chemiluminescence technique is widely used to measure ROS production by neutrophils; however, it is unclear which ROS species are detected by this technique. In this study, we show that Xanthine/Xanthine oxidase (XXO), a known superoxide-producing system, stimulated a luminol-amplified chemiluminescence in the absence of horseradish peroxidase (HRPO), while the presence of HRPO enhanced the response. Both reactions were inhibited by superoxide dismutase (SOD), but not by catalase, confirming that superoxide anion, and not hydrogen peroxide, is the species oxidizing luminol to produce chemiluminescence. Glucose/Glucose oxidase (GGO), a known hydrogen peroxide-producing system, did not induce luminol-amplified chemiluminescence in the absence of HRPO; however, addition of HRPO resulted in a chemiluminescence response, which was inhibited by catalase, but not by SOD. Myeloperoxidase (MPO), isolated from human neutrophils, was also able to enhance the superoxide- and hydrogen peroxide-dependent luminol-amplified chemiluminescence. The production of ROS by stimulated human neutrophils was detected by luminol-amplified chemiluminescence, which was only partially inhibited by SOD and catalase. Interestingly, adding HRPO to stimulated neutrophils increased the luminol-amplified chemiluminescence, which was strongly inhibited by SOD, but not by catalase. These results show that (a) luminol-amplified chemiluminescence is able to detect superoxide anion in the absence of peroxidases, but not hydrogen peroxide; (b) in the presence of peroxidases, luminol-amplified chemiluminescence is able to detect both superoxide anion and hydrogen peroxide; and (c) luminol-amplified chemiluminescence detects mainly superoxide anion produced by neutrophils, especially in the presence of HRPO.

Dendritic cells (DCs) are a heterogeneous population of professional antigen-presenting cells and are key cells of the immune system, acquiring different phenotypes in accordance with their localization during the immune response. A subset of inflammatory DCs is derived from circulating monocytes (Mo) and has a key role in inflammation and infection. The pathways controlling Mo-DC differentiation are not fully understood. Our objective was to investigate the possible role of nicotinamide adenine dinucleotide phosphate reduced form oxidases (NOXs) in Mo-DC differentiation. In this study, we revealed that Mo-DC differentiation was inhibited by NOX inhibitors and reactive oxygen species scavengers. We show that the Mo-DC differentiation was dependent on p22phox, and not on gp91phox/NOX2, as shown by the reduced Mo-DC differentiation observed in chronic granulomatous disease patients lacking p22phox. Moreover, we revealed that NOX5 expression was strongly increased during Mo-DC differentiation, but not during Mo-macrophage differentiation. NOX5 was expressed in circulating myeloid DC, and at a lower level in plasmacytoid DC. Interestingly, NOX5 was localized at the outer membrane of the mitochondria and interacted with p22phox in Mo-DC. Selective inhibitors and small interfering RNAs for NOX5 indicated that NOX5 controlled Mo-DC differentiation by regulating the JAK/STAT/MAPK and NFκB pathways. These data demonstrate that the NOX5-p22phox complex drives Mo-DC differentiation, and thus could be critical for immunity and inflammation.

Strait et al. described a novel mouse model of cryoglobulinaemia by challenging mice deficient in the immunoglobulin (Ig)G1 subclass (γ1(-) mice) with goat anti-mouse IgD [5]. The phenotype of wild-type mice was not remarkable, whereas γ1(-) mice developed IgG3 anti-goat IgG cryoglobulins as well as severe and lethal glomerulonephritis. Renal phenotype could not be rescued in γ1(-) mice by the deletion of C3, fragment crystalline γ receptor (FcγR) or J chain. On the other hand, early injection of IgG1, IgG2a or IgG2b inhibited the pathogenic effects of IgG3 in an antigen-dependent manner even in the absence of the FcγRIIb, an anti-inflammatory receptor. The authors concluded that the pathogenic role of IgG3 and the protective characteristic of IgG1 in this model were not explained by their abilities to bind to FcRs or effector molecules but are rather due to structural discrepancies enhancing the precipitation properties/solubility of IgG3/IgG1-containing immune complexes. The present article aims to discuss the current knowledge on IgG biology and the properties of IgGs explaining their differential propensity to acquire cryoglobulin activity.

Background & Aims In immune cells, constitutively and acutely produced type I interferons (IFNs) engage autocrine/paracrine signaling pathways to induce IFN-stimulated genes (ISGs). Enhanced activity of IFN signaling pathways can cause excessive inflammation and tissue damage. We aimed to investigate ISG expression in systemic immune cells from patients with decompensated alcoholic cirrhosis, and its association with outcome. Methods Peripheral blood mononuclear cells (PBMCs) from patients and heathy subjects were stimulated or not with lipopolysaccharide (LPS, an IFN inducer) or increasing concentrations of IFN-β. The expression of 48 ISGs and ten "non-ISG" inflammatory cytokines were analyzed using RT-qPCR. Results We developed an 8-ISG signature (IFN score) assessing ISG expression. LPS-stimulated ISG induction was significantly lower in PBMCs from patients with cirrhosis compared to healthy controls. Non-ISGs, however, showed higher induction. Lower induction of ISGs by LPS was not due to decreased IFN production by cirrhotic PBMCs or neutralization of secreted IFN, but a defective PBMC response to IFN. This defect was at least in part due to decreased constitutive ISG expression. Patients with the higher baseline IFN scores and ISG levels had the higher risk of death. At baseline, "non-ISG" cytokines did not correlate with outcome. Conclusions PBMCs from patients with decompensated alcoholic cirrhosis exhibit downregulated ISG expression, both constitutively and after an acute stimulus. Our finding that higher baseline PBMC ISG expression was associated with higher risk of death, suggests that constitutive ISG expression in systemic immune cells contributes to the prognosis of alcoholic cirrhosis. Lay summary Enhanced activity of IFN signaling pathways can cause excessive inflammation and tissue damage. Here we show that peripheral blood mononuclear cells (PBMCs) from patients with alcoholic cirrhosis exhibit a defect in interferon-stimulated genes (ISGs). We found that higher baseline ISG expression in PBMCs was associated with higher risk of death, revealing a probable contribution of ISG expression in immune cells to the outcome of alcoholic cirrhosis.

Adiponectin (Acrp30) belongs to the family of C1q/tumor necrosis factor α (TNFα)-related proteins. Acrp30 circulates as multimers of high, middle, and low molecular weight. In this study, we detected Acrp30 and its globular fragment (gAcrp30) in synovial fluid from rheumatoid arthritis patients. Intriguingly, the LMW form was more abundant in synovial fluid than in serum from both rheumatoid arthritis patients and healthy subjects. We also investigated the effects of Acrp30 and gAcrp30 on reactive oxygen species (ROS) production via the phagocytic NADPH oxidase. Acrp30 inhibited fMLF-induced ROS production by human phagocytes, whereas gAcrp30 enhanced it. gAcrp30's effect is additive with TNFα, whereas Acrp30 inhibited TNFα-induced priming. gAcrp30 enhanced NOX-2 expression at the plasma membrane, with a concomitant increase in p47(phox) phosphorylation. Selective inhibitors of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase 1 (ERK1)/2 abrogated p47(phox) phosphorylation by gAcrp30. In contrast, p47(phox) phosphorylation was inhibited by Acrp30 in association with increased AMP-activated protein kinase (AMPK) phosphorylation in phagocytes. These results suggest that human phagocyte ROS production is regulated by different mechanisms selective for Acrp30 versus gAcrp30. An imbalance between gAcrp30 and higher molecular weight isoforms of Acrp30 might contribute to chronic inflammation by regulating NADPH oxidase.

Background Anvillea garcinii Coss. & Durieu (Anv) plant is used as a traditional North African medicine against several diseases associated with inflammation. At inflammatory sites, reactive oxygen species (ROS) produced in excess by activated phagocyte NADPH oxidase (NOX2) can accentuate inflammatory responses. Thus, we investigated if Anv-water soluble polysaccharides could modulate primary human neutrophil oxidative burst in vitro. Methods Human neutrophils were isolated from fresh whole blood and O2 .- generation was measured by cytochrome c reduction assays. Western blots were used to analyse the translocation of PKC, p47phox (a key component of NOX2 activity) to neutrophil plasma membrane. Also, myeloperoxidase (MPO) release in the extracellular medium was studied by western blots. Flow cytometric analysis was used to detect CD11b membrane expression. Results Water soluble polysaccharides from Anv dose-dependently inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLF)- and phorbol myristate acetate (PMA)-induced O2 .- generation by human neutrophils. Moreover, Anv-polysaccharides strongly inhibited PMA-induced PKCβ and p47phox translocation to membranes and p47phox phosphorylation on Ser328, a main PKC target. In contrast, polysaccharides extract from Zygophyllum gaetulum plant, which is also used as a traditional North African medicine against inflammatory diseases, was ineffective on this PKCβ-p47phox pathway. Further, Anv inhibited important neutrophil degranulation markers corresponding to myeloperoxidase (MPO) release and CD11b membrane expression. Conclusion The process of down-regulating NADPH oxidase by polysaccharides extracts from Anv provides new insights into the mechanism of Anv’s anti-inflammatory actions. Electronic supplementary material The online version of this article (doi:10.1186/s12906-016-1411-7) contains supplementary material, which is available to authorized users.

OBJECTIVE The use of CCR5 inhibitors requires a tool to predict human immunodeficiency virus type 2 (HIV-2) tropism, as established in HIV-1. The aim of our study was to identify genotypic determinants of HIV-2 tropism located in the gp105 V3 loop. METHODS HIV-2 tropism phenotypic assays were performed on 53 HIV-2 clinical isolates using GFP expressing human osteosarcoma T4 [GHOST(3)] cell lines expressing CD4 and CCR5 or CXCR4 coreceptors. The gp105 V3 loop was sequenced and analyzed. RESULTS Thirty-four HIV-2 isolates were classified as R5, 7 as X4, and 12 as X4/R5 (dual). Substitution at residue 18 was always associated with a dual/X4 tropism (P < .00001). The following determinants were associated with dual/X4 tropism: a global net charge of more than +6 (P < .00001), V19K/R mutation (P < .00001), S22A/F/Y mutation (P < .002), Q23R mutation (P < .00001), and insertions at residue 24 (P < .00001), I25L/Y (P < .0004), R28K (P < .0004), and R30K (P < .014). These mutations were not found in R5 isolates, except R28K and R30K, which were detected in 4 and 5 R5 isolates, respectively. The 4 major genotypic determinants of dual/X4 tropism were mutation at residue 18, V19 K/R mutation, insertions at residue 24, and V3 global net charge. CONCLUSIONS We established a strong association between HIV-2 phenotypic tropism and V3-loop sequences, allowing for the prediction of R5- and/or X4-tropic viruses in HIV-2 infection.

Mouse bone marrow erythropoiesis is homeostatic, whereas after acute anemia, bone morphogenetic protein 4 (BMP4)–dependent stress erythropoiesis develops in the spleen. The aim of this work was to compare spleen stress erythropoiesis and bone marrow erythropoiesis in a mouse model of zymosan-induced generalized inflammation, which induces long-lasting anemia and to evaluate the ability of erythropoietin (Epo) injections to correct anemia in this setting. The effects of zymosan and/or Epo injections on erythroid precursor maturation and apoptosis, serum interferon-γ levels, hematologic parameters, and spleen BMP4 expression were analyzed, as well as the effect of zymosan on red blood cell half-life. We found that bone marrow erythropoiesis is suppressed by inflammation and does not respond to Epo administration, despite repression of erythroblast apoptosis. On the contrary, a robust erythropoietic response takes place in the spleen after Epo injections in both control and zymosan-induced generalized inflammation mice. This specific response implies Epo-mediated induction of BMP4 expression by F4/80+ spleen macrophages, proliferation of stress burst-forming units-erythroid, and increased number of spleen erythroblasts. It allows only partial recovery of anemia, probably because of peripheral destruction of mature red cells. It is not clear whether similar BMP4-dependent stress erythropoiesis can occur in human bone marrow after Epo injections.

Objective. The SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, and osteitis) is a rare disorder that mainly affects bone and skin. Chronic multifocal osteitis is the main diagnostic feature. Genetic studies of HLA genes have shown no role for these class II antigens, whereas studies of 2 mouse models (cmo and Lupo) point to a role of the PSTPIP2 gene. We analyzed the PSTPIP2 gene in patients with SAPHO syndrome.Methods. In a cohort of 38 patients with SAPHO we analyzed PSTPIP2 and 2 other candidate genes, NOD2/CARD15 (Crohn's disease occurs in about 10% of SAPHO patients), and LPIN2 (clinical similarities of SAPHO with Majeed syndrome).Results. Rare variants of the 3 genes observed in patients with SAPHO were not specific or were not found more frequently compared to controls, suggesting no major pathogenetic role of these genes in the SAPHO syndrome.Conclusion. We found no association between PSTPIP2, NOD2, and LPIN2 variants and the SAPHO syndrome.

The mechanisms linking severe inflammation and coagulation during heatstroke are poorly understood. Here, we examined the roles of the tissue factor pathway, leukocyte activation, and mediators of innate immunity in patients admitted to an intensive care unit for heatstroke during an intense heat wave in Paris.Retrospective observational study.Intensive care unit of a university medical center.Eighteen critically ill severe patients with heatstroke were enrolled in the study and 14 age-matched patients with severe sepsis as controls.None.High circulating levels of some inflammation and stress mediators (interleukin-6, -8, C5a, interleukin-1 receptor antagonist, heat shock protein 60 and 70) were observed. Blood leukocyte activation was shown by beta2 integrin up-regulation, L-selectin down-regulation, and strong production of reactive oxygen species and interleukin-8 ex vivo. High levels of circulating promatrix metalloproteinase-9 were detected in all the patients studied, and its active form was present in two patients. Overt disseminated intravascular coagulation according to the International Society of Thrombosis and Hemostasis score was present in five patients. Whole-blood tissue factor was present in all the patients and part of this activity was associated with microparticles in five patients. The degrees of inflammation and disseminated intravascular coagulation are correlated with clinical severity.These results suggest that neutrophil activation plays a key role in the acute activation of coagulation observed during severe heatstroke, despite a rapid and sustained antiinflammatory response. The comparison with a group of patients with severe sepsis suggests some common mechanisms, but more intense responses during heatstroke.

Introduction Le purpura rhumatoide (PR) est une maladie systemique caracterisee par la presence de depots d’IgA dans la peau, le rein et d’autres organes. Il n’existe aucun marqueur diagnostique et pronostique predictif de l’atteinte renale et de son evolution. Nous avons emis l’hypothese, basee sur les acquis de la nephropathie a IgA, que certaines cytokines, immunoglobulines, complexes immuns contenant des IgA (IgA–IgG, IgA–sCD89), IgA1 hypo-galactosylees (Gd-IgA1) et/ou NGAL pourraient distinguer les patients avec ou sans atteinte renale et identifier les patients a risque de progression vers l’insuffisance renale. Patients et methodes Cette etude multicentrique prospective a inclus 135 patients atteints de PR, evalues a la phase aigue de la maladie (atteinte cutanee active, j0) et apres un an de suivi (M12). Resultats A j0, dans le serum, les IgA, IgE, NGAL, IL-6, IL-8, IL-1β, les complexes IgA-IgG et IgA-sCD89 sont augmentees chez les patients par rapport aux temoins. Parmi les 135 patients, 93 (69 %) ont une atteinte renale (PRN). Les patients PRN ont une augmentation dans le serum des Gd-IgA1 et dans les urines des IgA, IgG, IgM, NGAL, IL-1β, IL-6, IL-8, IL-10, des complexes IgA-IgG et IgA-sCD89. A M12, 23 des 93 PRN ont evolue defavorablement (proteinurie/creatinine > 0,5 g/g, baisse du DFG ou deces). Les facteurs cliniques associes a cette evolution defavorable sont l’âge, le diabete, l’hypertension et la baisse du DFG. Les marqueurs biologiques sont dans les urines les IgA, les IgM, les complexes IgA-sCD89 et IgA-IgG. Ils sont capables de discriminer les patients de mauvais pronostic. Discussion Cette large cohorte prospective d’adultes et enfants atteints de PR, tous inclus a la phase aigue de la maladie, permet une meilleure comprehension de sa physiopathologie et confirme ses liens etroits avec la nephropathie a IgA. Le peu de patients evoluant defavorablement a un an limite notre etude. Le suivi de la cohorte va etre prolonge. Conclusion L’identification de biomarqueurs diagnostiques, capables de distinguer precocement les patients avec ou sans nephropathie et pronostiques, capables de predire l’evolution renale defavorable devraient ameliorer la prise en charge des patients atteints de PR en aidant les cliniciens et concepteurs d’essais cliniques a selectionner au mieux les patients les plus a meme de beneficier des futurs traitements innovants.

HIV-2 is naturally resistant to nonnucleoside reverse transcriptase inhibitors, to a fusion inhibitor, and to some of the protease inhibitors. Maraviroc is the first drug of the new anti-CCR5 drug class and is effective only on CCR5-tropic (R5) HIV-1. No previous studies concerning HIV-2 susceptibility to maraviroc have been reported yet. We developed a phenotypic maraviroc susceptibility test using a peripheral blood mononuclear cell (PBMC) model. We analyzed the maraviroc susceptibility of 13 R5 HIV-2, 2 X4R5 (dual) HIV-2, and 2 CXCR4-tropic (X4) HIV-2 clinical isolates. We also tested, with the same protocol, 1 X4 HIV-1 and 4 R5 HIV-1 clinical isolates. For the R5 HIV-2 clinical isolates, the 50% effective concentration (EC 50 ) for maraviroc was 0.80 nM (interquartile range [IQR], 0.48 to 1.39 nM), similar to that observed for the R5 HIV-1 isolates. The median maximum percentage of inhibition in the R5 HIV-2 isolates was 93% (IQR, 84 to 98%), similar to that observed in the R5 HIV-1 isolates. As expected, both X4 HIV-1 and HIV-2 were highly resistant to maraviroc. Our study showed for the first time that maraviroc is active in vitro against R5 HIV-2. The new tools we developed will allow identification of HIV-2-infected patients eligible for CCR5 inhibitor use and management of virological failure when receiving a maraviroc-based regimen.

Rheumatoid arthritis (RA), one of the most frequent chronic inflammatory rheumatic disorders, is characterized by the presence of autoantibodies and joint infiltration by activated immune cells, leading to cartilage and bone destruction. IgA occurs predominantly as monomers (mIgA) in plasma and regulates many cell responses through interaction with the Fcα receptor type I (FcαRI). FcαRI targeting by anti-FcαRI Fab inhibits activating receptors by inducing an inhibitory immunoreceptor tyrosine-based activation motif (ITAMi) configuration through SH2 domain-containing phosphatase 1 (SHP-1) recruitment. The aim of this study was to investigate the potential utility of mIgA for the treatment of arthritis by acting as an inducer of ITAMi signaling.The effect of plasma-derived human mIgA on inhibition of multiple heterologous receptors was evaluated on FcαRI+ cell transfectants, blood phagocytes from healthy individuals, and synovial cells from RA patients. FcαRI-transgenic mice and wild-type mice treated with mIgA were studied in models of collagen antibody-induced arthritis (CAIA) and collagen-induced arthritis (CIA). The mice were assessed for development of arthritis using an arthritis score, and joint tissue samples were evaluated for the extent of leukocyte infiltration and expression of phosphatase.Treatment with mIgA impaired cell activation in an FcαRI-FcRγ-dependent manner, involving ITAMi signaling. Human mIgA or anti-FcαRI Fab were strongly effective in either preventing or attenuating CAIA or CIA in FcαRI-transgenic mice. Administration of mIgA markedly inhibited the recruitment of leukocytes to the inflamed joints of mice, which was associated with induction of SHP-1 phosphorylation in joint tissue cells. Moreover, mIgA reversed the state of inflammation in the synovial fluid of RA patients by inducing an ITAMi configuration.These results demonstrate a therapeutic potential of human mIgA in experimental arthritis. The findings support future clinical exploration of mIgA for the treatment of RA.

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce reactive oxygen species (ROS) such as superoxide anion (O2 (∙-)), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl), a process referred to as the respiratory burst. The enzyme responsible for this process is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox), and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. The NADPH oxidase is in a resting state in circulating neutrophils, and its activation can be induced by a large number of soluble and particulate agents such as the formylated peptide, formyl-methionyl-leucyl-phenylalanine (fMLF). This activation can be enhanced or "primed" by pro-inflammatory cytokines, LPS and other agents. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS that can damage surrounding tissues and participate to inflammation. In this chapter, we describe the techniques used to measure priming of the NADPH oxidase in human neutrophils.

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce reactive oxygen species (ROS) such as superoxide anion (O2 (∙-)), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl), a process referred to as the respiratory burst. The enzyme responsible for this process is called the NADPH oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two transmembrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox), and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. The NADPH oxidase is in a resting state in circulating neutrophils, and its activation can be induced by a large number of soluble and particulate agents such as the formylated peptide, formyl-methionyl-leucyl-phenylalanine (fMLF). This activation can be enhanced or "primed" by pro-inflammatory cytokines, LPS and other agents. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS that can damage surrounding tissues and participate to inflammation. In this chapter, we describe the techniques used to measure priming of the NADPH oxidase in human neutrophils.

Superoxide anion production by the neutrophil NADPH oxidase plays a key role in host defense; however, excessive superoxide production is believed to participate to inflammatory reactions. Neutrophils express several TLR that recognize a variety of microbial motifs or agonists. The interaction between TLR and their agonists is believed to help neutrophils to recognize and eliminate the pathogen. However, the effects of some TLR agonists on the NADPH oxidase activation and the mechanisms controlling these effects have not been elucidated. In this study, we show that the TLR7/8 agonist CL097 by itself did not induce NADPH oxidase activation in human neutrophils, but induced a dramatic increase of fMLF-stimulated activation. Interestingly, CL097 induced cytochrome b558 translocation to the plasma membrane and the phosphorylation of the NADPH oxidase cytosolic component p47phox on Ser345, Ser328, and Ser315. Phosphorylation of Ser328 and Ser315 was significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser345, Ser328, and Ser315 was decreased by inhibitors of p38 MAPK and the ERK1/2 pathway. Phosphorylation of Ser328 was decreased by a protein kinase C inhibitor. Genistein, a broad-range protein tyrosine kinase inhibitor, inhibited the phosphorylation of these serines. Our results also show that CL097 induced proline isomerase 1 (Pin1) activation and that juglone, a Pin1 inhibitor, inhibited CL097-mediated priming of fMLF-induced p47phox phosphorylation and superoxide production. These results show that the TLR7/8 agonist CL097 induces hyperactivation of the NADPH oxidase by stimulating the phosphorylation of p47phox on selective sites in human neutrophils and suggest that p38 MAPK, ERK1/2, protein kinase C, and Pin1 control this process.

The pathophysiology of SAPHO syndrome still remains to be determined. However, like in other forms of spondylarthritides, this rare condition seems to result from the combination of genetic, environmental and immunological factors. Surely, SAPHO syndrome cannot be simply regarded as the adult form of the ‘caricatural’ DIRA (deficiency in interleukin-1 receptor antagonist) syndrome, although this purely genetic disease also causes multiple osteomyelitis and pustular rashes. An initial bacterial trigger, mainly represented by the cutaneous saprophyte Propionibacterium acnes, could take advantage of a selective deficiency of the innate immunity, implicating neutrophils. This could elicit thereafter a ‘hyperimmune’ reaction, as in other chronic inflammatory conditions like reactive arthritis, Crohn’s disease or hidradenitis suppurativa. The reported efficacy of either longterm antibiotic regimens (especially with azithromycin) or immunomodulatory biologic agents targetting TNF-α or IL-1 supports the concept of a post- or para-infectious hyperresponsiveness disorder, with a convincing rationale for ‘hybrid’ therapies.

Reactive oxygen species (ROS) production by the neutrophil NADPH oxidase plays a key role in host defense against pathogens, such as bacteria and fungi. Zymosan a cell-wall preparation from Saccharomyces cerevisiae is largely used to activate neutrophils in its opsonized form. In this study, we show that non-opsonized zymosan alone induced ROS production by human neutrophils. Zymosan-induced ROS production is higher than the formyl-methionyl-leucyl-phenylalanine (fMLF)- or the phorbol myristate acetate (PMA)-induced ROS production but is lower than the one induced by opsonized zymosan. Most of the zymosan-induced ROS production is intracellular. Interestingly, zymosan induced the phosphorylation of the NADPH oxidase cytosolic component p47phox on several sites which are Ser315, Ser328 and Ser345. Zymosan induced also the activation of the small G-protein Rac2. Phosphorylation of the p47phox as well as Rac2 activation were inhibited by genistein a broad range protein tyrosine kinase inhibitor and by wortmannin a PI3Kinase inhibitor. GF109203X a PKC inhibitor inhibited phosphorylation of p47phox on Ser315 and Ser328. SB203580 and UO126, inhibitors of p38MAPK and ERK1/2-pathway, respectively, inhibited phosphorylation of p47phox on Ser345. Zymosan-induced ROS production was completely inhibited by genistein and wortmannin and partially inhibited by SB203580, UO126 and GF109203X. These results show that zymosan alone is able to activate NADPH oxidase in human neutrophils via the phosphorylation of p47phox and Rac2 activation and that a protein tyrosine kinase, PI3Kinase, p38MAPK, ERK1/2 and PKC are involved in this process. These pathways could be potential pharmacological targets to treat zymosan- and S. cerevisiae-induced inflammation.

Background & Aims Lipopolysaccharide (LPS)-expressing bacteria cause severe inflammation in cirrhotic patients. The global gene response to LPS is unknown in cirrhotic immune cells. Methods Gene-expression profiling using Affymetrix Human Exon Array analyzed the expression of 14,851 genes in LPS-stimulated peripheral blood mononuclear cells (PBMCs) from 4 patients with cirrhosis and 4 healthy subjects. We performed validation studies using RT-qPCR in LPS-stimulated PBMCs from 52 patients and 9 healthy subjects and investigated the association of gene induction with mortality in 26 patients. Results Gene-expression profiling of LPS-stimulated cirrhotic cells showed 509 upregulated genes and 1588 downregulated genes. In LPS-stimulated "healthy" cells, 952 genes were upregulated and 838 genes downregulated. The 741 LPS-regulated genes shared by cirrhotic and "healthy" cells were involved in cytokine production/activity and induction of "immune paralysis". Comparison of functions associated with the 1356 genes, specifically regulated by LPS in cirrhotic cells, to functions of the 1049 genes, specifically regulated in "healthy" cells, allowed to define a cirrhosis-specific phenotype. Unlike in "healthy" cells, LPS failed to induce an interferon-mediated program in cirrhotic cells. In cirrhotic PBMCs, LPS specifically induced certain molecules involved in apoptosis and downregulated molecules involved in endocytic trafficking. RT-qPCR experiments showed that LPS-stimulated cirrhotic PBMCs had an enhanced induction of certain proinflammatory cytokines and chemokines. In the prognosis study, higher ex vivo LPS-induction of the inflammatory genes IL6 and CXCL5 was a significant predictor of mortality. Conclusions Our results show that LPS-stimulated cirrhotic PBMCs exhibit an extensive and often unexpected transcriptional response.

BACKGROUND Only a few studies based on small cohorts have been carried out on iron status in anorexia nervosa (AN) patients. OBJECTIVE The aim of this study was to evaluate the role of hepcidin in hyperferritinemia in AN adolescents. DESIGN Twenty-seven adolescents hospitalized for AN in the pediatric inpatient unit of Ambroise Pare Academic Hospital were enrolled in the study. The control group comprised 11 patients. Hematologic variables and markers of iron status, including serum hepcidin, were measured before and after nutritional rehabilitation. RESULTS The mean age of patients was 14.4 y. Except for 2 AN patients and 1 control patient, all patients presented normal hemoglobin, vitamin B-12, and folate concentrations. Markers of inflammation and cytokines were normal throughout the study. None of the muscular lysis markers were elevated. Most AN patients had normal serum iron concentrations on admission. Serum ferritin concentrations were significantly higher in patients than in control subjects (198 compared with 49 μg/L, respectively; P < 0.001). The median hepcidin concentration was significantly higher in AN patients than in the control group (186.5 compared with 39.5 μg/L, respectively; P = 0.002). There was a highly significant correlation between ferritinemia and serum hepcidin concentrations (P < 0.0001). After nutritional rehabilitation, a significant reduction was observed (P = 0.004) in serum ferritin. Serum hepcidin analyzed in a smaller number of patients also returned to within the normal range. CONCLUSIONS Hepcidin and ferritin concentrations were higher in the serum of AN patients, without any evidence of iron overload or inflammation. These concentrations returned to normal after nutritional rehabilitation. These results suggest that nutritional stress induced by malnourishment in the hepatocyte could be yet another mechanism that regulates hepcidin.