Outcome of SARS-CoV-2 infection is linked to MAIT cell activation and cytotoxicity.

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.

ROCK2 interacts with p22phox to phosphorylate p47phox and to control NADPH oxidase activation in human monocytes.

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.

Impaired p47phox phosphorylation in neutrophils from patients with p67phox-deficient chronic granulomatous disease.

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.

Blood fibrocytes are associated with severity and prognosis in COVID-19 pneumonia.

Increased blood fibrocytes are associated with a poor prognosis in fibrotic lung diseases. We aimed to determine whether the percentage of circulating fibrocytes could be predictive of severity and prognosis during coronavirus disease 2019 (COVID-19) pneumonia. Blood fibrocytes were quantified by flow cytometry as CD45+/CD15-/CD34+/collagen-1+ cells in patients hospitalized for COVID-19 pneumonia. In a subgroup of patients admitted in an intensive care unit (ICU), fibrocytes were quantified in blood and bronchoalveolar lavage (BAL). Serum amyloid P (SAP), transforming growth factor-ß1 (TGF-ß1), CXCL12, CCL2, and FGF2 concentrations were measured. We included 57 patients in the hospitalized group (median age = 59 yr [23-87]) and 16 individuals as healthy controls. The median percentage of circulating fibrocytes was higher in the patients compared with the controls (3.6% [0.2-9.2] vs. 2.1% [0.9-5.1], P = 0.04). Blood fibrocyte count was lower in the six patients who died compared with the survivors (1.6% [0.2-4.4] vs. 3.7% [0.6-9.2], P = 0.02). Initial fibrocyte count was higher in patients showing a complete lung computed tomography (CT) resolution at 3 mo. Circulating fibrocyte count was decreased in the ICU group (0.8% [0.1-2.0]), whereas BAL fibrocyte count was 6.7% (2.2-15.4). Serum SAP and TGF-ß1 concentrations were increased in hospitalized patients. SAP was also increased in ICU patients. CXCL12 and CCL2 were increased in ICU patients and negatively correlated with circulating fibrocyte count. We conclude that circulating fibrocytes were increased in patients hospitalized for COVID-19 pneumonia, and a lower fibrocyte count was associated with an increased risk of death and a slower resolution of lung CT opacities.

Priming of the neutrophil respiratory burst: role in host defense and inflammation.

Neutrophils are the major circulating white blood cells in humans. They play an essential role in host defense against pathogens. In healthy individuals, circulating neutrophils are in a dormant state with very low efficiency of capture and arrest on the quiescent endothelium. Upon infection and subsequent release of pro-inflammatory mediators, the vascular endothelium signals to circulating neutrophils to roll, adhere, and cross the endothelial barrier. Neutrophils migrate toward the infection site along a gradient of chemo-attractants, then recognize and engulf the pathogen. To kill this pathogen entrapped inside the vacuole, neutrophils produce and release high quantities of antibacterial peptides, proteases, and reactive oxygen species (ROS). The robust ROS production is also called 'the respiratory burst', and the NADPH oxidase or NOX2 is the enzyme responsible for the production of superoxide anion, leading to other ROS. In vitro, several soluble and particulate agonists induce neutrophil ROS production. This process can be enhanced by prior neutrophil treatment with 'priming' agents, which alone do not induce a respiratory burst. In this review, we will describe the priming process and discuss the beneficial role of controlled neutrophil priming in host defense and the detrimental effect of excessive neutrophil priming in inflammatory diseases.

NOX5 and p22phox are 2 novel regulators of human monocytic differentiation into dendritic cells.

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.

Macrophage Polarization Favors Epithelial Repair During Acute Respiratory Distress Syndrome.

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.

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

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.

Value of biomarkers for predicting immunoglobulin A vasculitis nephritis outcome in an adult prospective cohort.

Background: Henoch-Schönlein 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)-1ß, 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 < 0.005 for all comparisons). After follow-up (1 year), 22 patients showed a poor outcome. Among the tested markers, urine IgA at disease onset adequately reclassified the risk of poor outcome over conventional clinical factors, including estimated glomerular filtration rate, proteinuria and age (continuous net reclassification improvement = 0.72, P = 0.001; integrated discrimination improvement = 0.13, P = 0.009) in IgAV patients. Conclusions: Taken together, these results showed that serum Gd-IgA1 and urinary IgA, IgG, IgM, NGAL, IL-1ß, IL-6, IL-8, IL-10, IgA-IgG and IgA-sCD89 complexes were associated with nephritis in IgAV patients. Urinary IgA level may improve patient risk stratification for poor outcome.

Type I interferon signaling in systemic immune cells from patients with alcoholic cirrhosis and its association with outcome.

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-ß. 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.

Protective role of mouse IgG1 in cryoglobulinaemia; insights from an animal model and relevance to human pathology.

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.

Anvillea garcinii extract inhibits the oxidative burst of primary human neutrophils.

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 PKCß 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 PKCß-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.

Pulmonary manifestations in adult patients with chronic granulomatous disease.

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.

Identification of secreted phosphoprotein 1 gene as a new rheumatoid arthritis susceptibility gene.

OBJECTIVE: To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the rheumatoid arthritis (RA) genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. METHODS: We analysed a total of 11,715 RA cases and 26,493 controls from nine independent cohorts; all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on osteopontin (OPN) expression in macrophages and OPN serum levels was investigated. RESULTS: We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (p=1.29×10(-5), ORACPA-negative 1.257 (1.135 to 1.394)) and less with ACPA positivity (p=0.0148, ORACPA-positive 1.072 (1.014 to 1.134)). The ORs between these subgroups (ie, ACPA-positive and ACPA-negative) significantly differed (p=7.33×10(-3)). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (p=0.0157), as well as serum level of secreted OPN correlated positively with ACPA production (p=0.005; r=0.483). CONCLUSIONS: We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association being greater in patients negative for ACPAs.

The TLR7/8 agonist CL097 primes N-formyl-methionyl-leucyl-phenylalanine-stimulated NADPH oxidase activation in human neutrophils: critical role of p47phox phosphorylation and the proline isomerase Pin1.

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 Ser(345), Ser(328), and Ser(315). Phosphorylation of Ser(328) and Ser(315) was significantly increased in CL097-primed and fMLF-stimulated neutrophils. Phosphorylation of Ser(345), Ser(328), and Ser(315) was decreased by inhibitors of p38 MAPK and the ERK1/2 pathway. Phosphorylation of Ser(328) 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.

Gene- and exon-expression profiling reveals an extensive LPS-induced response in immune cells in patients with cirrhosis.

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.

Adiponectin and its globular fragment differentially modulate the oxidative burst of primary human phagocytes.

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.

Increased reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorders patients with JAK2 (V617F) mutation.

Myeloproliferative disorders are associated with increased risk of thrombosis and vascular complications. The pathogenesis of these complications is not completely known. Reactive oxygen species produced by the neutrophil NADPH oxidase could have a role in this process. The aim of this study was to evaluate reactive oxygen species production by neutrophils of myeloproliferative disorder patients. Patients with or without the JAK2 V617F mutation were characterized. Reactive oxygen species production was assessed by chemiluminescence, and phosphorylation of the NADPH oxidase subunit p47phox was analyzed by Western blots. In a comparison of controls and myeloproliferative disorder patients without the JAK2 V617F mutation, reactive oxygen species production by neutrophils from patients with the JAK2 V617F mutation was dramatically increased in non-stimulated and in stimulated conditions. This increase was associated with increased phosphorylation of the p47phox on Ser345 and of the uspstream kinase ERK1/2. In neutrophils from healthy donors, JAK2 can be activated by GM-CSF. GM-CSF-induced p47phox phosphorylation and priming of reactive oxygen species production are inhibited by the selective JAK2 inhibitors AG490 and lestaurtinib (CEP-701), supporting a role for JAK2 in the upregulation of NADPH oxidase activation. These findings show an increase in reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorder patients with the JAK2 V617F mutation, and demonstrate that JAK2 is involved in GM-CSF-induced NADPH oxidase hyperactivation. As neutrophil hyperactivation could be implicated in the thrombophilic status of patients with myeloproliferative disorders, aberrant activation of JAK2 V617F, leading to excessive neutrophil reactive oxygen species production might play a role in this setting.

Molecular determinants of HIV-2 R5-X4 tropism in the V3 loop: development of a new genotypic tool.

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.