Reactive Oxygen Species and Neutrophil Function.

Neutrophils are essential for killing bacteria and other microorganisms, and they also have a significant role in regulating the inflammatory response. Stimulated neutrophils activate their NADPH oxidase (NOX2) to generate large amounts of superoxide, which acts as a precursor of hydrogen peroxide and other reactive oxygen species that are generated by their heme enzyme myeloperoxidase. When neutrophils engulf bacteria they enclose them in small vesicles (phagosomes) into which superoxide is released by activated NOX2 on the internalized neutrophil membrane. The superoxide dismutates to hydrogen peroxide, which is used by myeloperoxidase to generate other oxidants, including the highly microbicidal species hypochlorous acid. NOX activation occurs at other sites in the cell, where it is considered to have a regulatory function. Neutrophils also release oxidants, which can modify extracellular targets and affect the function of neighboring cells. We discuss the identity and chemical properties of the specific oxidants produced by neutrophils in different situations, and what is known about oxidative mechanisms of microbial killing, inflammatory tissue damage, and signaling.

Intestinal IL-17R Signaling Controls Secretory IgA and Oxidase Balance in Citrobacter rodentium Infection.

Type 17 cytokines have been strongly implicated in mucosal immunity, in part by regulating the production of antimicrobial peptides. Using a mouse model of Citrobacter rodentium infection, which causes colitis, we found that intestinal IL-17RA and IL-17RC were partially required for control of infection in the colon and IL-17 regulates the production of luminal hydrogen peroxide as well as expression of Tnsf13 Reduced Tnfsf13 expression was associated with a profound defect in generating C. rodentium-specific IgA+ Ab-secreting cells. Taken together, intestinal IL-17R signaling plays key roles in controlling invading pathogens, in part by regulating luminal hydrogen peroxide as well as regulating the generation of pathogen-specific IgA+ Ab-secreting cells.

Defense of Scots pine against sawfly eggs (Diprion pini) is primed by exposure to sawfly sex pheromones.

Plants respond to insect infestation with defenses targeting insect eggs on their leaves and the feeding insects. Upon perceiving cues indicating imminent herbivory, such as damage-induced leaf odors emitted by neighboring plants, they are able to prime their defenses against feeding insects. Yet it remains unknown whether plants can amplify their defenses against insect eggs by responding to cues indicating imminent egg deposition. Here, we tested the hypothesis that a plant strengthens its defenses against insect eggs by responding to insect sex pheromones. Our study shows that preexposure of Pinus sylvestris to pine sawfly sex pheromones reduces the survival rate of subsequently laid sawfly eggs. Exposure to pheromones does not significantly affect the pine needle water content, but results in increased needle hydrogen peroxide concentrations and increased expression of defense-related pine genes such as SOD (superoxide dismutase), LOX (lipoxygenase), PAL (phenylalanine ammonia lyase), and PR-1 (pathogenesis related protein 1) after egg deposition. These results support our hypothesis that plant responses to sex pheromones emitted by an herbivorous insect can boost plant defensive responses to insect egg deposition, thus highlighting the ability of a plant to mobilize its defenses very early against an initial phase of insect attack, the egg deposition.

Neutrophils suppress mucosal-associated invariant T cells in humans.

Mucosal-associated invariant T (MAIT) cells are innate-like T lymphocytes that are abundant in mucosal tissues and the liver where they can respond rapidly to a broad range of riboflavin producing bacterial and fungal pathogens. Neutrophils, which are recruited early to sites of infection, play a nonredundant role in pathogen clearance and are crucial for controlling infection. The interaction of these two cell types is poorly studied. Here, we investigated both the effect of neutrophils on MAIT cell activation and the effect of activated MAIT cells on neutrophils. We show that neutrophils suppress the activation of MAIT cells by a cell-contact and hydrogen peroxide dependent mechanism. Moreover, highly activated MAIT cells were able to produce high levels of TNF-α that induced neutrophil death. We therefore provide evidence for a negative regulatory feedback mechanism in which neutrophils prevent overactivation of MAIT cells and, in turn, MAIT cells limit neutrophil survival.

Efficacy of two adjuvants administrated with a novel hydrogen peroxide-inactivated vaccine against Streptococcus agalactiae in Nile tilapia fingerlings.

Streptococcus agalactiae is considered the main bacterial pathogen in cultured Nile tilapia. Formaldehyde-inactivated vaccines are the most accepted method for prevention and control of the disease. However, alternative inactivation methods for S. agalactiae vaccines have not been fully explored. Recently, we developed a hydrogen peroxide-inactivated vaccine against S. agalactiae with moderate efficacy, with the possibility to improve vaccine efficacy by adding adjuvants. The current study compared the efficacy of aluminum hydroxide and Freund's incomplete adjuvant (FIA) incorporated into a novel hydrogen peroxide-inactivated intraperitoneal vaccine against S. agalactiae for Nile tilapia fingerlings. The relative percentage survival (RPS) for aluminum hydroxide-adjuvanted vaccine (59.3%), and FIA-adjuvanted vaccine (77.8%) were higher than the vaccine without adjuvant (40.7%). In addition, fish immunized with aluminum hydroxide-adjuvanted vaccine had significantly higher levels of specific antibodies than control fish at 4 weeks post vaccination (wpv). Blood lymphocytes counts showed a decrease in vaccinated groups when compared to control fish, suggesting white cells migration to the tissues where antigen presentation is ongoing. Fish that received FIA-adjuvanted vaccine exhibited persistence of adjuvant deposits on intraperitoneal surfaces for at least 4 wpv that may be related to its superior performance compared to aluminum hydroxide adjuvanted vaccine, which did not evidence any type of deposit at any sampling times. The results observed in this study demonstrate that hydrogen peroxide-inactivated vaccine administered with either aluminum hydroxide or FIA induce optimal levels of protection, with a superior performance for FIA vaccine, which could be a good alternative to conventional formaldehyde-inactivated vaccines against S. agalactiae, due to its shorter manufacture time, and less toxicity.

Methyl jasmonate induces the resistance of postharvest blueberry to gray mold caused by Botrytis cinerea.

BACKGROUND: The effects of postharvest methyl jasmonate (MeJA) treatment (50 µmol L-1 ) on the control of gray mold caused by Botrytis cinerea in blueberry fruit were evaluated by analyzing (i) the levels of disease resistance signals; (ii) the activity of enzymes involved in antioxidant system, disease resistance and phenylpropanoid pathway, and (iii) the secondary metabolite content. RESULTS: The results indicated that MeJA treatment significantly restrained the development of gray mold decay in blueberries. The treatment induced a nitric oxide (NO) burst and increased the endogenous hydrogen peroxide (H2 O2 ) content in the earlier period of storage. The enhanced NO and H2 O2 generation by MeJA treatment might serve as a signal to induce resistance against B. cinerea infection. Furthermore, in inoculated fruit, MeJA treatment significantly promoted antioxidant enzymes and defense-related enzyme activity, which included superoxide dismutase, catalase, ascorbate peroxidase, chitinase, and ß-1,3-glucanase, and the degree of membrane lipid peroxidation was reduced. The MeJA treatment enhanced the phenylpropanoid pathway by provoking phenylalanine ammonialyase, cinnamate 4-hydroxylase, and 4-coumarate CoA ligase activity, which was accompanied by elevated levels of phenolics and flavonoids in blueberry fruit. CONCLUSION: These results suggested that MeJA could induce the disease resistance of blueberries against B. cinerea by regulating the antioxidant enzymes, defense-related enzymes, and the phenylpropanoid pathway through the activation of signaling molecules.

Sensitization potential and potency of terpene hydroperoxides in the cocultured activation test method.

BACKGROUND: Positive patch test reactions to mixtures of oxidized terpenes containing allergenic hydroperoxides are frequently reported. However, human sensitization data for these hydroperoxides are not available. OBJECTIVES: To analyse and evaluate the human sensitization potential and potency of hydroperoxides in vitro by using human cells. MATERIALS/METHODS: Limonene-1-hydroperoxide, limonene-2-hydroperoxide, citronellol-7-hydroperoxide, cumene hydroperoxide, 1-(1-hydroperoxy-1-methylethyl)cyclohexene and mixtures of citronellol hydroperoxides (isomers at positions 6 and 7) and linalool hydroperoxides (isomers at positions 6 and 7) were studied. All compounds were synthesized except for cumene hydroperoxide, which was commercially available. Their potential and potency to activate dendritic cells (DCs) was evaluated by measuring the upregulation of CD86 and CD54 on THP-1 cells upon exposure in the cocultured activation test (COCAT) consisting of HaCaT cells (human keratinocyte cell line) and THP-1 monocytes (as a surrogate for DCs). RESULTS: Hydroperoxides upregulated CD86 and/or CD54 on cocultured THP-1 cells in a concentration-dependent manner. The results are comparable with their sensitization potency ranking in predictive animal models. CONCLUSIONS: For the first time, the human sensitization potential and potency of several hydroperoxides were determined by the use of human cells and the COCAT method.

Self-Supplied Tumor Oxygenation through Separated Liposomal Delivery of H2O2 and Catalase for Enhanced Radio-Immunotherapy of Cancer.

The recent years have witnessed the blooming of cancer immunotherapy, as well as their combinational use together with other existing cancer treatment techniques including radiotherapy. However, hypoxia is one of several causes of the immunosuppressive tumor microenvironment (TME). Herein, we develop an innovative strategy to relieve tumor hypoxia by delivering exogenous H2O2 into tumors and the subsequent catalase-triggered H2O2 decomposition. In our experiment, H2O2 and catalase are separately loaded within stealthy liposomes. After intravenous (iv) preinjection of CAT@liposome, another dose of H2O2@liposome is injected 4 h later. The sustainably released H2O2 could be decomposed by CAT@liposome, resulting in a long lasting effect in tumor oxygenation enhancement. As the result, the combination treatment by CAT@liposome plus H2O2@liposome offers remarkably enhanced therapeutic effects in cancer radiotherapy as observed in a mouse tumor model as well as a more clinically relevant patient-derived xenograft tumor model. Moreover, the relieved tumor hypoxia would reverse the immunosuppressive TME to favor antitumor immunities, further enhancing the combined radio-immunotherapy with cytotoxic T lymphocyte-associated antigen 4 (CTLA4) blockade. This work presents a simple yet effective strategy to promote tumor oxygenation via sequential delivering catalase and exogenous H2O2 into tumors using well-established liposomal carriers, showing great potential for clinical translation in radio-immunotherapy of cancer.

Oxidative stress drives CD8+ T-cell skin trafficking in patients with vitiligo through CXCL16 upregulation by activating the unfolded protein response in keratinocytes.

BACKGROUND: In patients with vitiligo, an increased reactive oxygen species (ROS) level has been proved to be a key player during disease initiation and progression in melanocytes. Nevertheless, little is known about the effects of ROS on other cells involved in the aberrant microenvironment, such as keratinocytes and the following immune events. CXCL16 is constitutively expressed in keratinocytes and was recently found to mediate homing of CD8+ T cells in human skin. OBJECTIVE: We sought to explicate the effect of oxidative stress on human keratinocytes and its capacity to drive CD8+ T-cell trafficking through CXCL16 regulation. METHODS: We first detected putative T-cell skin-homing chemokines and ROS in serum and lesions of patients with vitiligo. The production of candidate chemokines was detected by using quantitative real-time PCR and ELISA in keratinocytes exposed to H2O2. Furthermore, the involved mediators were analyzed by using quantitative real-time PCR, Western blotting, ELISA, and immunofluorescence. Next, we tested the chemotactic migration of CD8+ T cells from patients with vitiligo mediated by the CXCL16-CXCR6 pair using the transwell assay. RESULTS: CXCL16 expression increased and showed a positive correlation with oxidative stress levels in serum and lesions of patients with vitiligo. The H2O2-induced CXCL16 expression was due to the activation of 2 unfolded protein response pathways: kinase RNA (PKR)-like ER kinase-eukaryotic initiation factor 2α and inositol-requiring enzyme 1α-X-box binding protein 1. CXCL16 produced by stressed keratinocytes induced migration of CXCR6+CD8+ T cells derived from patients with vitiligo. CXCR6+CD8+ T-cell skin infiltration is accompanied by melanocyte loss in lesions of patients with vitiligo. CONCLUSION: Our study demonstrated that CXCL16-CXCR6 mediates CD8+ T-cell skin trafficking under oxidative stress in patients with vitiligo. The CXCL16 expression in human keratinocytes induced by ROS is, at least in part, caused by unfolded protein response activation.

Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity.

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca(2+) entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H2O2 scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca(2+) concentration that did not increase further in response to H2O2. Additionally, ΔkatG-infected macrophages displayed limited Ca(2+) influx in response to ionomycin, as a result of ionophore H2O2 sensitivity. Exogenously added H2O2 or H2O2 generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca(2+). Basal increases in cytosolic Ca(2+) and insensitivity to H2O2-mediated Ca(2+) entry in ΔkatG-infected cells are reversed by the Ca(2+) channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H2O2-mediated Ca(2+) entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca(2+), actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca(2+) signaling and limit host immune function.

Slc11a1 (Nramp-1) gene modulates immune-inflammation genes in macrophages during pristane-induced arthritis in mice.

OBJECTIVE AND DESIGN: Pristane-induced arthritis (PIA) in AIRmax mice homozygous for Slc11a1 R and S alleles was used to characterize the influence of Slc11a1 gene polymorphism on immune responses during disease manifestation. Previous reports demonstrated that the presence of the Slc11a1 S allele increased the incidence and severity of PIA in AIRmax SS , suggesting that this gene could interact with inflammatory loci to modulate PIA. We investigated the effects of Slc11a1 alleles on the activation of phagocytes during PIA. TREATMENT: Mice were injected intraperitoneally with two doses of 0.5 mL of mineral oil pristane at 60-day intervals. Arthritis development was accompanied for 180 days. RESULTS: AIRmax SS mice showed differential peritoneal macrophage gene expression profiles during PIA, with higher expression and production of H2O2, NO, IL-1ß, IL-6, TNF-α, and several chemokines. The presence of the Slc11a1 R allele, on the other hand, diminished the intensity of macrophage activation, restricting arthritis development. CONCLUSION: Our data demonstrated the fine-tuning roles of Slc11a1 alleles modulating macrophage activation, and consequent PIA susceptibility, in those mouse lines.

Melatonin is required for H2 O2 - and NO-mediated defense signaling through MAPKKK3 and OXI1 in Arabidopsis thaliana.

Melatonin influences plant innate immunity through the mitogen-activated protein kinase (MAPK) pathway. However, the most upstream MAPK component in melatonin signaling and the dependence of generation of a reactive oxygen species (ROS) burst on melatonin synthesis and signaling remain unclear. In this study, treatment of several mekk (alias mapkkk)-knockout Arabidopsis mutants with melatonin revealed that the MAPKKK3 and OXI1 (oxidative signal-inducible1) kinases are responsible for triggering melatonin-induced defense signaling pathways. In addition, melatonin induction upon infection with the avirulent pathogen Pseudomonas syringae DC3000 (avrRpt2) was independent of H2 O2 and NO individually, but dependent on the combination of H2 O2 and NO. Moreover, melatonin-mediated induction of the expression of defense-related genes, such as PR1 and ICS1, was not altered in the H2 O2 -deficient rbohD/F-knockout mutant cotreated with an NO scavenger, indicating that melatonin functions downstream of the ROS and NO burst. Collectively, the data indicate that melatonin-mediated induction of an innate immune response requires multiple signaling molecules and activation of MAPKKK3 and OXI1, followed by triggering of downstream MAPK cascades, such as MAPK3 and MAPK6.

Duox1-derived H2O2 modulates Cxcl8 expression and neutrophil recruitment via JNK/c-JUN/AP-1 signaling and chromatin modifications.

DUOX1-derived hydrogen peroxide (H2O2) and CXCL8 are two key neutrophil chemoattractants. H2O2 is critical at the early phase, whereas CXCL8 plays a key role in the late phases of recruitment, but the crosstalks between the two phases in vivo remain unknown. In this study using zebrafish, we report that H2O2 also contributes to neutrophil recruitment to injuries at the late phase as it induces Cxcl8 expression in vivo through a JNK/c-JUN/AP-1 signaling pathway. However, Erk and NF-κB signaling were not involved in this crosstalk. Strikingly, H2O2 also promotes cxcl8 expression through modulation of histone 3 lysine 4 trimethylation, histone 3 lysine 9 acetylation, and histone 3 lysine 9 trimethylation levels at its promoter. These results explain how early H2O2 signal regulates neutrophil recruitment at all phases, directly via Lyn oxidation or indirectly by modulating cxcl8 gene expression, via the activation of redox-sensitive signaling pathways, and further point out H2O2/DUOX1 as a key drug target for anti-inflammatory therapies.

Contact-dependent depletion of hydrogen peroxide by catalase is a novel mechanism of myeloid-derived suppressor cell induction operating in human hepatic stellate cells.

Myeloid-derived suppressor cells (MDSC) represent a unique cell population with distinct immunosuppressive properties that have been demonstrated to shape the outcome of malignant diseases. Recently, human hepatic stellate cells (HSC) have been reported to induce monocytic-MDSC from mature CD14(+) monocytes in a contact-dependent manner. We now report a novel and unexpected mechanism by which CD14(+)HLADR(low/-) suppressive cells are induced by catalase-mediated depletion of hydrogen peroxide (H2O2). Incubation of CD14(+) monocytes with catalase led to a significant induction of functional MDSC compared with media alone, and H2O2 levels inversely correlated with MDSC frequency (r = -0.6555, p < 0.05). Catalase was detected in primary HSC and a stromal cell line, and addition of the competitive catalase inhibitor hydroxylamine resulted in a dose-dependent impairment of MDSC induction and concomitant increase of H2O2 levels. The NADPH-oxidase subunit gp91 was significantly increased in catalase-induced MDSC as determined by quantitative PCR outlining the importance of oxidative burst for the induction of MDSC. These findings represent a so far unrecognized link between immunosuppression by MDSC and metabolism. Moreover, this mechanism potentially explains how stromal cells can induce a favorable immunological microenvironment in the context of tissue oxidative stress such as occurs during cancer therapy.

The receptor-like cytoplasmic kinase BSR1 mediates chitin-induced defense signaling in rice cells.

Broad-Spectrum Resistance 1 (BSR1) encodes a rice receptor-like cytoplasmic kinase, and enhances disease resistance when overexpressed. Rice plants overexpressing BSR1 are highly resistant to diverse pathogens, including rice blast fungus. However, the mechanism responsible for this resistance has not been fully characterized. To analyze the BSR1 function, BSR1-knockout (BSR1-KO) plants were generated using a clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Experiments using suspension-cultured cells revealed that defense responses including H2O2 production (i.e. oxidative burst) and expression of defense-related genes induced by autoclaved conidia of the rice blast fungus significantly decreased in BSR1-KO cells. Furthermore, a treatment with chitin oligomers which function as microbe-associated molecular patterns (MAMPs) of the rice blast fungus resulted in considerably suppressed defense responses in BSR1-KO cells. These results suggest that BSR1 is important for the rice innate immunity triggered by the perception of chitin.

ATP modulates acute inflammation in vivo through dual oxidase 1-derived H2O2 production and NF-κB activation.

Dual oxidase 1 (Duox1) is the NADPH oxidase responsible for the H2O2 gradient formed in tissues after injury to trigger the early recruitment of leukocytes. Little is known about the signals that modulate H2O2 release from DUOX1 and whether the H2O2 gradient can orchestrate the inflammatory response in vivo. In this study, we report on a dominant-negative form of zebrafish Duox1 that is able to inhibit endogenous Duox1 activity, H2O2 release and leukocyte recruitment after tissue injury, with none of the side effects associated with morpholino-mediated Duox1 knockdown. Using this specific tool, we found that ATP release following tissue injury activates purinergic P2Y receptors, and modulates Duox1 activity through phospholipase C (PLC) and intracellular calcium signaling in vivo. Furthermore, Duox1-derived H2O2 is able to trigger the NF-κB inflammatory signaling pathway. These data reveal that extracellular ATP acting as an early danger signal is responsible for the activation of Duox1 via a P2YR/PLC/Ca(2+) signaling pathway and the production of H2O2, which, in turn, is able to modulate in vivo not only the early recruitment of leukocytes to the wound but also the inflammatory response through activation of the NF-κB signaling pathway.

CD36-mediated hematoma absorption following intracerebral hemorrhage: negative regulation by TLR4 signaling.

Promoting hematoma absorption is a novel therapeutic strategy for intracerebral hemorrhage (ICH); however, the mechanism of hematoma absorption is unclear. The present study explored the function and potential mechanism of CD36 in hematoma absorption using in vitro and in vivo ICH models. Hematoma absorption in CD36-deficient ICH patients was examined. Compared with patients with normal CD36 expression, CD36-deficient ICH patients had slower hematoma adsorption and aggravated neurologic deficits. CD36 expression in perihematomal tissues in wild-type mice following ICH was increased, whereas the hematoma absorption in CD36(-/-) mice was decreased. CD36(-/-) mice also showed aggravated neurologic deficits and increased TNF-α and IL-1ß expression levels. The phagocytic capacity of CD36(-/-) microglia for RBCs was also decreased. Additionally, the CD36 expression in the perihematoma area after ICH in TLR4(-/-) and MyD88(-/-) mice was significantly increased, and hematoma absorption was significantly promoted, which was significantly inhibited by an anti-CD36 Ab. In vitro, TNF-α and IL-1ß significantly inhibited the microglia expression of CD36 and reduced the microglia phagocytosis of RBCs. Finally, the TLR4 inhibitor TAK-242 upregulated CD36 expression in microglia, promoted hematoma absorption, increased catalase expression, and decreased the H2O2 content. These results suggested that CD36 mediated hematoma absorption after ICH, and TLR4 signaling inhibited CD36 expression to slow hematoma absorption. TLR4 inhibition could promote hematoma absorption and significantly improve neurologic deficits following ICH.

Air-oxidized linalyl acetate - an emerging fragrance allergen?

BACKGROUND: Linalyl acetate is a fragrance chemical that is prone to autoxidation. Exposure to linalyl acetate occurs through cosmetic products and essential oils, but is difficult to assess, as linalyl acetate is not labelled in the EU. OBJECTIVE: To investigate the frequencies of contact allergy to oxidized linalyl acetate among dermatitis patients, and to investigate the autoxidation of linalyl acetate in terms of hydroperoxide formation and sensitization potency. PATIENTS AND METHODS: Hydroperoxide formation in air-exposed linalyl acetate was determined with high-performance liquid chromatography. The sensitization potencies of hydroperoxides were determined with the local lymph node assay. One thousand seven hundred and seventeen patients were patch tested with oxidized linalyl acetate at 6.0% in petrolatum. RESULTS: Of the patients, 2.2% showed positive reactions to oxidized linalyl acetate. Forty-three per cent of the positive patients also had positive patch test reactions to other fragrance markers. Linalyl acetate hydroperoxides were detected early in the autoxidation process, and accumulated to a concentration of 37% after 42 weeks of air exposure. The linalyl acetate hydroperoxides were classified as moderate sensitizers. CONCLUSIONS: The frequency of positive reactions to oxidized linalyl acetate is comparable to that of previously studied oxidized fragrance terpenes. Oxidized linalyl acetate could thus be a common fragrance contact allergen.

FcγR-driven release of IL-6 by macrophages requires NOX2-dependent production of reactive oxygen species.

Activation of the FcγR via antigen containing immune complexes can lead to the generation of reactive oxygen species, which are potent signal transducing molecules. However, whether ROS contribute to FcγR signaling has not been studied extensively. We set out to elucidate the role of NADPH oxidase-generated ROS in macrophage activation following FcγR engagement using antigen-containing immune complexes. We hypothesized that NOX2 generated ROS is necessary for propagation of downstream FcγR signaling and initiation of the innate immune response. Following exposure of murine bone marrow-derived macrophages (BMDMs) to inactivated Francisella tularensis (iFt)-containing immune complexes, we observed a significant increase in the innate inflammatory cytokine IL-6 at 24 h compared with macrophages treated with Ft LVS-containing immune complexes. Ligation of the FcγR by opsonized Ft also results in significant ROS production. Macrophages lacking the gp91(phox) subunit of NOX2 fail to produce ROS upon FcγR ligation, resulting in decreased Akt phosphorylation and a reduction in the levels of IL-6 compared with wild type macrophages. Similar results were seen following infection of BMDMs with catalase deficient Ft that fail to scavenge hydrogen peroxide. In conclusion, our findings demonstrate that ROS participate in elicitation of an effective innate immune in response to antigen-containing immune complexes through FcγR.

Antibacterial efficacy of recombinant Siganus oramin L-amino acid oxidase expressed in Pichia pastoris.

Siganus oraminl-amino acid oxidase is a novel natural protein (named SR-LAAO) isolated from serum of the rabbitfish (S. oramin), which showed antibacterial activity against both Gram-positive and Gram-negative bacteria and had a lethal effect on the parasites Cryptocaryon irritans, Trypanosoma brucei brucei and Ichthyophthirius multifiliis. In order to test whether recombinant SR-LAAO (rSR-LAAO) produced by the eukaryotic expression system also has antimicrobial activity, the yeast Pichia pastoris was used as the expression host to obtain rSR-LAAO in vitro. Crude rSR-LAAO produced by P. pastoris integrated with the SR-LAAO gene had antibacterial activity against both Gram-positive and Gram-negative bacteria as shown by inhibition zone assay of the antibacterial spectrum on agar plates. The average diameter of the inhibition zone of crude rSR-LAAO against the Gram-positive bacteria Staphylococcus aureus and Streptococcus agalactiae was 1.040 ± 0.045 cm and 1.209 ± 0.085 cm, respectively. For the Gram-negative bacteria Aeromonas sobria, Escherichia coli, Vibrio alginolyticus, Vibrio cholera and Photobacterium damselae subsp. piscicida, the average diameter of inhibition zone was 1.291 ± 0.089 cm, 0.943 ± 0.061 cm, 0.756 ± 0.057 cm, 0.834 ± 0.023 cm and 1.211 ± 0.026 cm, respectively. These results were obtained at the logarithmic growth phase of S. agalactiae and A. sobria cell suspensions after incubation with 0.5 mg/mL crude rSR-LAAO for 24 h. The final bacterial growth rate was decreased significantly. The relative inhibition rate can reach 50% compared to crude products from P. pastoris integrated with an empty vector at the same concentration of protein. The antimicrobial activity of crude rSR-LAAO was likely associated with H2O2 formation, because its inhibition zones were disturbed significantly by catalase. Scanning electron microscopy results showed crude rSR-LAAO-treated bacterial surfaces became rough and particles were attached, cell walls were retracted and cell membranes were ruptured. Together, the results of this study indicated rSR-LAAO from the P. pastoris expression system is a potential antibiotic for application as a therapeutic agent against bacterial diseases.