Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function

Phagocytosis is a fundamental cellular process that is pivotal for immunity as it coordinates microbial killing, innate immune activation and antigen presentation. An essential step in this process is phagosome acidification, which regulates a number of functions of these organelles that allow them to participate in processes essential to both innate and adaptive immunity. Here we report that acidification of phagosomes containing Gram-positive bacteria is regulated by the NLRP3-inflammasome and caspase-1. Active caspase-1 accumulates on phagosomes and acts locally to control the pH by modulating buffering by the NADPH oxidase NOX2. These data provide insight into a mechanism by which innate immune signals can modify cellular defenses and establish a new function for the NLRP3-inflammasome and caspase-1 in host defense

High-Dose Mannose-Binding Lectin Therapy for Ebola Virus Infection

Mannose-binding lectin (MBL) targets diverse microorganisms for phagocytosis and complement-mediated lysis by binding specific surface glycans. Although recombinant human MBL (rhMBL) trials have focused on reconstitution therapy, safety studies have identified no barriers to its use at higher levels. Ebola viruses cause fatal hemorrhagic fevers for which no treatment exists and that are feared as potential biothreat agents. We found that mice whose rhMBL serum concentrations were increased ≥7-fold above average human levels survived otherwise fatal Ebola virus infections and became immune to virus rechallenge. Because Ebola glycoproteins potentially model other glycosylated viruses, rhMBL may offer a novel broad-spectrum antiviral approach

Lectin-Dependent Enhancement of Ebola Virus Infection via Soluble and Transmembrane C-type Lectin Receptors

Mannose-binding lectin (MBL) is a key soluble effector of the innate immune system that recognizes pathogen-specific surface glycans. Surprisingly, low-producing MBL genetic variants that may predispose children and immunocompromised individuals to infectious diseases are more common than would be expected in human populations. Since certain immune defense molecules, such as immunoglobulins, can be exploited by invasive pathogens, we hypothesized that MBL might also enhance infections in some circumstances. Consequently, the low and intermediate MBL levels commonly found in human populations might be the result of balancing selection. Using model infection systems with pseudotyped and authentic glycosylated viruses, we demonstrated that MBL indeed enhances infection of Ebola, Hendra, Nipah and West Nile viruses in low complement conditions. Mechanistic studies with Ebola virus (EBOV) glycoprotein pseudotyped lentiviruses confirmed that MBL binds to N-linked glycan epitopes on viral surfaces in a specific manner via the MBL carbohydrate recognition domain, which is necessary for enhanced infection. MBL mediates lipid-raft-dependent macropinocytosis of EBOV via a pathway that appears to require less actin or early endosomal processing compared with the filovirus canonical endocytic pathway. Using a validated RNA interference screen, we identified C1QBP (gC1qR) as a candidate surface receptor that mediates MBL-dependent enhancement of EBOV infection. We also identified dectin-2 (CLEC6A) as a potentially novel candidate attachment factor for EBOV. Our findings support the concept of an innate immune haplotype that represents critical interactions between MBL and complement component C4 genes and that may modify susceptibility or resistance to certain glycosylated pathogens. Therefore, higher levels of native or exogenous MBL could be deleterious in the setting of relative hypocomplementemia which can occur genetically or because of immunodepletion during active infections. Our findings confirm our hypothesis that the pressure of infectious diseases may have contributed in part to evolutionary selection of MBL mutant haplotypes

MBL mediates HIV-EBOV GP infection via the canonical macropinocytosis pathway for EBOV but with less dependence on actin.

<p>We preincubated HEK293F cells with (A) EIPA (5-(<i>N</i>-Ethyl-<i>N</i>-isopropyl)amiloride, a potent and specific inhibitor of Na⁺/H⁺ exchanger activity), (B) methyl-β-cyclodextrin (extracts or sequesters cholesterol from the plasma membrane), (C) latrunculin B (blocks actin polymerization), (D) cytochalasin D (inhibits actin microfilament function), (E) nocodazole (disrupts microtubules), or (F) jasplakinolide (disrupts microtubules) in 5% MBL-deficient serum in the absence or presence of rhMBL at 37°C for 1 hour. We then infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl). Percentages of infected cells are relative to DMSO controls. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate. Significant differences are shown. (G) Absorbance values of an ELISA assay are shown indicating the difference in amount of rhMBL within the physiological range that binds to immobilized mannan or FITC-dextran (1 µg/100 µl). (H) We preincubated FITC-dextran with various concentrations of rhMBL at 37°C for 30 minutes and then added the products to PMA-stimulated (10 ng/ml), IL-4-supplemented (100 ng/ml) THP-1 cells at 37°C for 1 hour. We measured FITC-dextran uptake by flow cytometry and reported the results as mean fluorescence intensity (geometric mean fluorescence × percentage of cells). Experiments were performed twice in triplicate.</p

MBL interacts with HIV-EBOV GP via MBL carbohydrate recognition domains.

<p>We preincubated 5% serum containing native human MBL (3,621 ng/ml) with (A) 0, 1 and 10 mM of hexose monosaccharides or EDTA diluted in media, or (B) 0–100 µg/ml of mannan or polydisperse polyethylene glycol (PEG)(D) at room temperature for 30 minutes. Then we incubated the serum with HIV-EBOV GP (1200 pg p24/100 µl) at 37°C for 1 hour before infecting adherent HEK293F cells. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). We observed relatively more toxicity associated with 10 mM EDTA but this did not invalidate our results because of our adjustment for cell viability. (C) We repeated the previous experiments with 3F8, an anti-human MBL monoclonal antibody or an IgG1 isotype control (preincubation at 37°C for 30 minutes). Significant differences are shown. (D) We preincubated HIV-EBOV GP virion-like particles with cyanovirin (0–600 nM) at 37°C for 1 hour before incubating the particles with 5% serum in the presence or absence of rhMBL. Luciferase values were adjusted for cell viability. Experiments were performed twice in quadruplicate.</p

MBL targets <i>N</i>-linked glycans on viral and cellular surfaces.

<p>The cleavage sites of two endoglycosidases are shown (A,B). N-glycosidase F (PNGase F) is an amidase that cleaves the linkages between the innermost GlcNAc and asparagine residues within high-mannose, hybrid and complex oligosaccharides of <i>N</i>-linked glycoproteins, thereby producing carbohydrate-free peptides without any potential ligands for MBL. Endoglycosidase H (endo H) cleaves linkages within the diacetylchitobiose stem of high-mannose of <i>N</i>-linked glycoproteins, thereby generating a truncated sugar molecule with one <i>N</i>-acetylglucosamine residue (a potential target for MBL) remaining on the asparagine. Man, mannose; GlcNAc, <i>N</i>-acetylglucosamine; asn, asparagine; × and y, various oligosaccharides; n = 2–150 residues. We preincubated HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) at 37°C for 1 hour with (C) PNGase F or endo H (0–10,000 U/ml), or (D) the same concentrations of heat inactivated enzymes. Then we incubated the viruses with 5% MBL-deficient serum in the presence or absence of rhMBL at 37°C for 1 hour before infecting HEK293F cells. Significant differences are shown. (E) We preincubated HEK293F cells at 37°C for 1 hour with chemicals (tunicamycin, swainsonine or deoxynojirimycin) that inhibit various stages of <i>N</i>-linked glycosylation. Then we infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) that had been preincubated with 5% MBL-deficient serum and supplemented with various concentrations of rhMBL. Significant differences are shown. * and **, p<0.001 (all pairwise comparisons at 1 and 10 µg/ml rhMBL, respectively). (F) We cultivated HEK293F and HEK293S (deficient in <i>N</i>-acetylglucosaminyltransferase I) cells in 5% MBL-deficient serum which was supplemented with various concentrations of rhMBL. We infected cells with HIV-EBOV-GP virion-like particles (1200 pg p24/100 µl) in the absence or presence of 1 µg/ml tunicamycin. Statistical differences among inhibitors at various rhMBL concentrations are shown. *, ** and †, p<0.005 (all pairwise comparisons at 0.1, 1 and 10 µg/ml rhMBL, respectively). Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay). All experiments were performed twice in quadruplicate.</p

MBL enhances HIV-EBOV GP infection of THP-1 cells and human monocyte-derived macrophages.

<p>(A) We stimulated 5×10⁴ THP-1 cells with PMA (10 ng/ml) and supplemented the cells with IL-4 (100 ng/ml) for 72 hours. We preincubated HIV-EBOV GP or HIV-<i>env</i> negative virion-like particles (1200 pg p24/100 µl) with or without rhMBL before infecting differentiated adherent THP-1 cells cultivated in 5% MBL-deficient serum. (B) We cultivated 2.5×10⁵ PBMC derived from human single-donor buffy coat samples in RPMI-1640 with 10% FBS and stimulated the cells with M-CSF (50 ng/ml) to induce differentiation of monocyte-derived macrophages. We infected cells with HIV-EBOV GP (WT), HIV-EBOV-ΔGP NTDL6 (NTDL6, mutated GP lacks 217 amino acids in the heavily glycosylated mucin-rich region) or HIV-<i>env</i> negative (env neg) in the presence or absence of rhMBL. The box plot represents outliers (dots), 10^th and 90^th percentiles (whiskers), 25^th and 75^th percentiles (box) and median values (line). Significant differences in infection rates are shown. Luciferase values were adjusted for cell viability using alamarBlue (resazurin reduction assay) for all the above experiments, which were performed twice in quadruplicate.</p