Neutrophil Oxidant Production Research Articles

Dysregulation of neutrophil oxidant production and interleukin-1-related cytokines in granulomatosis with polyangiitis.

Neutrophils play a key role in ANCA-associated vasculitis, both as targets of autoimmunity and as facilitators of vascular damage. In granulomatosis with polyangiitis (GPA), the data regarding the production of reactive oxygen species (ROS) in neutrophils are unclear. Further, recent data suggests that ROS production could have an anti-inflammatory effect through the regulation of inflammasomes and IL-1-related cytokines. We aimed to analyse ROS production in neutrophils from patients with GPA and investigate its association with IL-1-related cytokines and the autoantigen PR3. Seventy-two GPA patients with disease flare were included in the NEUTROVASC prospective cohort study. ROS production in whole blood of patients with active GPA was evaluated and compared with that in the same patients in remission or healthy controls. Associations between ROS production, PR3 membrane expression on neutrophils, serum levels of IL-1-related cytokines as well as inflammasome-related proteins were analysed. We observed a robust defect in ROS production by neutrophils from patients with active GPA compared with healthy controls, independent of glucocorticoid treatment. Serum levels of IL-1-related cytokines were significantly increased in GPA patients, particularly in patients with kidney involvement, and levels of these cytokines returned to normal after patients achieved remission. Further, inflammasome-related proteins were significantly dysregulated in the cytosol of neutrophils as well as the serum from GPA patients. Our data suggests that ROS production and regulation of inflammasomes in neutrophils from patients with GPA are disturbed and may be a potential therapeutic target. ClinicalTrials.gov, https://www.clinicaltrials.gov, NCT01862068.

Neutrophils and their friends.

Neutrophils and their friends.

Fibrotic lung disease inhibits immune responses to staphylococcal pneumonia via impaired neutrophil and macrophage function.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by collagen deposition within the lung interstitium. Bacterial infection is associated with increased morbidity and more rapid mortality in IPF patient populations, and pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are commonly isolated from the lungs of hospitalized patients with IPF. Despite this, the effects of fibrotic lung injury on critical immune responses to infection remain unknown. In the present study, we show that, like humans with IPF, fibrotic mice infected with MRSA exhibit increased morbidity and mortality compared with uninfected fibrotic mice. We determine that fibrosis conferred a defect in MRSA clearance compared with nonfibrotic mice, resulting from blunted innate immune responses. We show that fibrosis inhibited neutrophil intracellular killing of MRSA through impaired neutrophil elastase release and oxidative radical production. Additionally, we demonstrate that lung macrophages from fibrotic mice have impaired phagocytosis of MRSA. Our study describes potentially novel impairments of antimicrobial responses upon pulmonary fibrosis development, and our findings suggest a possible mechanism for why patients with IPF are at greater risk of morbidity and mortality related to infection.

<p>Regulation of NO production by MAPK dual-specificity phosphatases (DUSP) in human neutrophils exposed to N-nitrosodimethylamine</p>

One of the enzymes responsible for nitric oxide (NO) production in neutrophils is the inducible nitric oxide synthase (iNOS). Changes in its expression may result from the activation of different signaling pathways, including MAPK, which lead to activation of various genes, including DUSP genes. DUSP induce the negative feedback loop leading to MAPK deactivation through their phosphorylation. Our study assessed the role of DUSP1, DUSP10 and DUSP16 with the participation of MAPK in the iNOS-dependent NO production by neutrophils exposed to xenobiotic, N-nitrosodimethylamine (NDMA). The obtained results suggest that N-nitrosodimethylamine enhances the expression of all tested proteins (except DUSP10) in the cytoplasmic and nuclear fractions of neutrophils. The JNK pathway inhibition resulted in an extenuation of iNOS, phospho-p38 and DUSP10 expression in the cytoplasmic fraction and DUSP1 expression in the nuclear fraction of neutrophils. Inhibition of the p38 pathway led to a lower expression of iNOS, DUSP16 and DUSP10 in the cytoplasmic fraction. No changes in the phospho-JNK and DUSP1 expressions were observed. With the results of this study we can conclude that DUSP are positive regulators of MAP kinases in NDMA-induced signaling pathway which lead to modulation of iNOS-dependent NO production in human neutrophils

G-CSF Inhibits Growths of Osteoblasts and Osteocytes by Upregulating Nitric Oxide Production in Neutrophils.

Granulocyte colony-stimulating factor (G-CSF) is the critical regulator of the proliferation, differentiation, and survival of granulocytes. Recently, it has been shown that G-CSF can adversely affect bone health in both animal models and patients. Here, the authors aimed to investigate whether G-CSF could inhibit the growth of osteoblasts and osteocytes by regulating nitric oxide. The C57BL/6 mice were divided into the control group, G-CSF treatment group and recovery group (G-CSF+L-NAME). The morphology of femurs was assessed by histology and immunohistochemistry. The expression of apoptosis-related molecules in femurs was detected by immunohistochemistry and quantitative RT-PCR, respectively. To examine if neutrophil-secreted factors can induce apoptosis in osteoblasts, Gr1-positive (Gr1+) neutrophils from the bone marrow of wild-type mice were sorted and co-cultured with MC3T3 pre-osteoblasts for 2 days. The number of osteoblasts and newly embedding osteocytes significantly decreased and markers related to osteoblasts and osteocytes were downregulated in the G-CSF treatment compared to the control group. Moreover, G-CSF treatment did not change proliferation markers but induced apoptosis in osteoblast-lineage cells. The combined treatment of mice with G-CSF and a nitric oxide inhibitor partially restored the number of osteoblasts and osteocyte parameters. The G-CSF can inhibit osteoblasts and osteocytes by upregulating nitric oxide.

Effects of alpha-ketoglutarate (AKG) on growth performance and non-specific immunity of juvenile red drum fed diets with low or adequate phosphorus levels.

This study evaluated the effects of α-ketoglutarate (AKG) on the growth performance, body, and skeletal composition, as well as non-specific immunity of juvenile red drum (Sciaenops ocellatus) fed adequate or low-phosphorus diets. A 2 × 2 factorial design was arranged with two levels of total phosphorus (1.0 or 1.6%) and two levels (0 or 1%) of AKG. Each diet was fed to red drum in four replicate 110-L aquaria (15 fish/aquarium) twice daily for 7weeks. The results indicated that the low-P diets significantly (P< 0.05) decreased weight gain, feed efficiency, and condition factor of red drum, but increased moisture content and decreased ash content of whole body, as well as decreased ash content of skeletal tissue and decreased plasma lysozyme. Supplementation of AKG significantly improved the fillet yield and plasma lysozyme and tended to improve neutrophil oxidative radical production (P = 0.097). Based on these results, phosphorus level had the greatest effect on growth performance and non-specific immunity of red drum, while AKG supplementation had limited positive effects on immunological responses and fillet yield of juvenile red drum.

Opioid-mediated Neutrophil Microparticle Production and NLRP3 Inflammasome Activation Cause Vascular Damage

Background Opioid-receptors are found on leukocytes within and outside the central nervous system. Inflammation and interleukin (IL)-1β, in particular, play major roles in addiction. Because IL-1β must be packaged within extracellular vesicles for transport, we hypothesized that opioids stimulate production of microparticles (MPs). Methods Human and murine neutrophil MPs production were assessed by flow cytometry in response to morphine and hydromorphone ex vivo , and mice were injected with drugs to assess in vivo responses. Results Human and murine neutrophils generate MPs high in IL-1β by a complex oxidative stress response involving mitochondria, NADPH oxidase and nitric oxide synthase-2, similar to that seen with other stimuli (FRBM 106:406, 2017). After 1 hour incubation of 550 murine neutrophils, 0, 0.1, 1, 5 and 10 µM morphine triggered generation of, respectively (mean + SE, n=3, *p + 5, 296 + 34, 1351 + 179*, 2560 + 413* and 3113 + 982* MPs. IL-1β content of MPs was similarly increased such that control MPs had 1.6 + 0.6 pg/million MPs whereas those generated due to 10 µM morphine had 92.8 + 8.1 (p . 01) pg/million MPs. Morphine responses were inhibited by 1 µM naloxone (opioid-receptor antagonist). When injected into mice, morphine and hydromorphone increase blood-borne MPs with surface proteins indicative of production by neutrophils (Ly6G+), microglia (P2Y12 and CD45+) and endothelium (CD31+/CD41-dim). Time-course and dose-responses demonstrated diffuse capillary leak similar to that seen with other stimuli (J Appl Physiol 123:297, 2017) likely due to neutrophil MPs, spinal microglia activation assessed as CD68 expression by immunohistochemistry, and progressive elevations of circulating microglia MPs with repetitive dosing. Conclusion Opioid-receptor stimulation triggers neutrophil oxidative stress, MPs production and NLRP3 inflammasome activation. Circulating MPs cause vascular injuries and may play a critical role in opioid addiction.

The effects of environmental salinity on the growth and physiology of totoaba Totoaba macdonaldi and shortfin corvina Cynoscion parvipinnis.

Totoaba Totoaba macdonaldi and shortfin corvina Cynoscion parvipinnis, were acclimated and reared together at salinities of 0, 2, 5, 10, 20 and 35 for 56 days. Initial overall mean ± s.d. body masses of 67·6 ± 7·1 g T. macdonaldi and 37·3 ± 3·1 g C. parvipinnis increased to final overall masses of 217·4 ± 30·3 and 96·5 ± 16·5 g, respectively, at the end of the study. Totoaba macdonaldi was not able to tolerate salinities of 0 and 2 and C. parvipinnis of 0. In contrast, both species had 100% survival at salinities ≥ 10. Somatic growth was highest not at natural seawater salinity of 35, but at 10. Plasma osmolality ranged from 172·5 to 417·0 mOsmol kg-1 for T. macdonaldi and from 207·0 to 439·5 mOsmol kg-1 for C. parvipinnis and varied in direct proportion to salinity. The estimated isosmotic salinities of T. macdonaldi and C. parvipinnis were 12·3 and 13·4, respectively. Cynoscion parvipinnis reared at two had significantly lower plasma lysozyme activity (95·0 Units ml-1 ) than fish held at salinities from 5 to 35 (ranging from 215·0 to 355·0 Units ml-1 ), but without clear trends over this range. Blood neutrophil oxidative radical production (NBT) (ranging from 3·9 to 6·7 mg ml-1 ) had some significant differences among salinities, but these did not follow a clear pattern. For T. macdonaldi, neither lysozyme activity nor NBT was affected by salinity. Ash content of whole fishes varied directly and moisture content inversely, with salinity for both species.

Performance of Cage‐Raised, Overwintered Hybrid Striped Bass Fed Fish Meal‐ or Soybean‐Based Diets

AbstractWe conducted a 26‐week study to evaluate the performance of cage‐raised hybrid Striped Bass (female White Bass Morone chrysops × male Striped Bass M. saxatilis) fed fish meal (FM) and soybean (SOY; soybean meal + soy protein concentrate) based diets. A FM reference diet was formulated to contain crude protein (CP) at 450 g/kg, lipid at 120 g/kg, and an estimated digestible energy level of 13 MJ/kg. Two test diets (SOY and SOY+GBA) were formulated to replace 70% of the CP in the reference diet with SBM in the absence or presence of GroBiotic‐A (GBA), a yeast‐based prebiotic, at 20 g/kg. The other 12% of dietary FM in the reference diet was replaced with SPC in the test diets, further reducing FM inclusion to 70 g/kg. Each diet was fed to quadruplicate groups of 40 advanced juvenile hybrid Striped Bass (initial weight ∼ 54 g/fish) stocked into each of twelve 1‐m3 floating cages, which were equally divided into four 0.05‐ha, rubber‐lined ponds according to a randomized complete block design. Caged fish in each pond were fed one of the randomly assigned diets to apparent satiation once daily. After 26 weeks of feeding, results showed that (1) the overall growth performance, survival, and feed efficiency of the fish were unaffected by diet; (2) the intraperitoneal fat ratio was significantly lower in fish that received the SOY‐based diets; and (3) plasma glucose and lysozyme activity were significantly lower in fish that were fed the SOY‐based diets, whereas plasma osmolality, hematocrit, neutrophil oxidative radical production (nitroblue tetrazolium test), and extracellular superoxide anion production of head‐kidney macrophages were unaffected by diet. We conclude that low‐FM, SOY‐based diets can support adequate performance and normal physiological responses of hybrid Striped Bass during overwintering.

Neutrophil Activation In Sickle Cell Disease: Biochemical and Functional Changes At Baseline and During Acute Vaso-Occlusive Crises

Neutrophilia is a well-recognized complication of sickle cell disease (SCD) and is a risk factor for increased disease severity and early death. Increased neutrophil activation, adhesion and oxidant production have been reported in SCD. While changes in neutrophil surface marker expression, degranulation, migration, adhesion and intracellular oxidative stress have been reported in SCD, there is little mechanistic underpinning for these observed functional changes. Moreover, how neutrophils specifically contribute to SCD pathology, particularly during acute vaso-occlusive crises, remains unknown. We hypothesize that chronic biochemical and functional changes of neutrophils in SCD contribute to disease pathology and are amplified during vaso-occlusive crisis. To define biochemical differences between neutrophils isolated from individuals with SCD and those isolated from healthy controls, we performed global gene expression analysis using the Affymetrix HG133 plus 2 GeneChip. Highly distinct transcriptional profiles were observed in neutrophils isolated from controls versus individuals with SCD at baseline and SCD in crisis. These were sufficiently distinct as to be separable by disease group in unsupervised hierarchical clustering analyses. Ontological analyses identified enriched transcriptional networks in SCD neutrophils related to elevated reactive oxygen species (ROS) production (p<6.3E-4) as well as potentiated respiratory burst (p<0.008). Using extracellular flux technology, we were able to measure oxygen consumption rates (OCR) of intact cells and observed that neutrophils from individuals with SCD have an elevated basal OCR over control cells. The elevated basal OCR could be due to increased NADPH oxidase 2 activity or increased mitochondrial respiration. While mitochondrial activity in healthy neutrophils is minimal, these organelles are essential to drive apoptosis, which is required for adequate resolution of inflammatory processes. In turn, neutrophil apoptosis is suppressed in multiple inflammatory diseases, including SCD. The transcriptional studies identified an upregulation of genes that promote mitochondrial stability (2.1- to 3.5-fold increase, p<0.02), a potential mechanism for both increased neutrophil lifespan and perhaps ROS production. Normally, neutrophils maintain ample antioxidant capacity to prevent intracellular damage from ROS generation. Vitamin E is a particularly important antioxidant in neutrophil biology as it attenuates multiple pro-inflammatory activities of these cells. We find that SCD neutrophils have markedly diminished vitamin E levels (p=0.031). Thus, loss of this important antioxidant may promote a pro-inflammatory phenotype of neutrophils in SCD. Next, we examined the potential contribution of neutrophils to the SCD pathobiology in vivo particularly during acute crisis. Berkeley sickle mice (SS mice) and controls (AA mice) were exposed to normoxia (baseline) or hypoxia (3 hrs 10% FIO2) to induce acute sickling followed by reoxygenation (2 hrs room air, H/R). Liver histopathology of SS mice displayed areas of leukocyte infiltration at baseline, with a potentiation in the number of these inflammatory sites after acute sickling induced by H/R. Importantly, examination of these sites at higher magnification reveals significant neutrophil infiltration near congested vessels, suggesting that H/R may potentiate neutrophil recruitment to injured tissues in SCD. To further assess neutrophil activation post H/R, plasma levels of myeloperoxidase (MPO) and DNA, evidence of neutrophil extracellular trap (NET) formation, were measured. SS mice have elevated plasma MPO levels compared to AA mice at baseline (p=0.007), with a trend towards a further increase in SS mice post H/R (no change in plasma MPO in AA mice post H/R). Plasma DNA levels were also elevated in SS mice compared to AA mice at baseline (p=0.01) and exposure of SS mice to H/R significantly increased plasma DNA levels (p=0.009), consistent with an acute increase in NET formation. In summary, chronic neutrophil activation in SS mice at baseline is exacerbated by acute sickling. Taken together, our data supports a role for neutrophils in SCD pathology which is amplified during vaso-occlusive crisis. Thus, targeting neutrophils may offer a novel therapeutic strategy for the prevention and treatment of vaso-occlusive complications in SCD.▪ Disclosures:Wandersee:Bayer: Consultancy. Hillery:Bayer: Consultancy; Biogen Idec: Consultancy.

Effect of progesterone receptor status on maspin synthesis via nitric oxide production in neutrophils in human breast cancer

Although progesterone receptor (PR) status, similarly to estrogen receptor status, is of prognostic importance in breast cancer, the involvement of the PR in breast cancer remains obscure. Studies were conducted to determine the function of the PR in neutrophils in the nitric oxide-induced synthesis of maspin, an anti-breast-cancer protein produced in nonmalignant mammary cells and in neutrophils in the circulation. PR status was determined by immunohistochemistry. Maspin synthesis was determined by in-vitro translation of messenger RNA and quantified by enzyme-linked immunosorbent assay. Nitric oxide was determined by the methemoglobin method. It was found that PR status in neutrophils was identical with that in malignant breast tissues. A Scatchard plot for progesterone binding to normal and PR-positive (PR+) neutrophils revealed that whereas normal neutrophils had 11.5×10(10) PR sites/cell with K d=47.619nM, PR+ neutrophils had 6.6×10(10) PR sites/cell with K d=47.619nM. The progesterone negative (PR-) neutrophils failed to bind to progesterone. Incubation of normal and PR+ neutrophils with 25nM progesterone produced 1.317μM NO and 2.329nM maspin; the PR+ neutrophils produced 0.72μM NO and 1.138nM maspin. The PR- neutrophils failed to produce any NO or maspin in the presence of progesterone. Inhibition of progesterone-induced NO synthesis led to complete inhibition of maspin synthesis in all neutrophils. These results suggest that estrogen and progesterone complement each other in NO-induced maspin synthesis, and do not necessarily antagonize in the synthesis of the anti-breast-cancer protein.

Dietary supplementation of arginine and/or glutamine influences growth performance, immune responses and intestinal morphology of hybrid striped bass (Morone chrysops × Morone saxatilis)

Various studies with fish species indicate that dietary arginine (Arg) and glutamine (Gln) supplementation may support an enhanced performance beyond simply improving weight gain. Therefore, a feeding trial was conducted to determine the effects of dietary Arg or Gln on growth performance, immune responses and intestinal morphology of juvenile hybrid striped bass, Morone chrysops×Morone saxatilis (4.1±0.02g, initial average weight). The basal diet was formulated from menhaden fishmeal and solvent-extracted soybean meal to contain 45% crude protein and 10% lipid. Arginine and Gln were singularly added to the basal diet at either 1% or 2% or both amino acids were added in combination at 1% to formulate experimental diets with adjustments in glycine to maintain all diets iso-nitrogenous. Fish were fed the experimental diets for a period of 8weeks, at the end of which final weight, specific growth rate and feed efficiency were significantly (P<0.05) higher in fish fed diets supplemented with Arg at either 1 or 2%, Gln at 1%, Arg and Gln both at 1%, but not in those fed the diet supplemented with Gln at 2%.Arginine and/or Gln supplementation tended to improve neutrophil oxidative radical production, but not significantly (P>0.05). However, significantly higher serum lysozyme activity was observed in fish fed the diet supplemented with Arg at either 1 or 2% and Gln at 1%. Intracellular superoxide anion production by hybrid striped bass macrophages also tended to increase, while extracellular superoxide anion production was significantly improved by supplementation of Arg at 1% and Gln at either 1 or 2% of diet.Morphometric analyses demonstrated positive effects of both dietary Arg and Gln on histo-morphological measurements in different portions of the gastrointestinal tract of hybrid striped bass. The 1% Arg diet resulted in the greatest increases in size of the chosen enteric structures, but the other four experimental diets also significantly improved the scores for various intestinal portions compared to fish fed the basal diet.Results from the present experiment demonstrate the importance of dietary supplementation of both Arg and Gln in improving growth performance, eliciting positive changes to several components of the innate immune system, and also benefiting the intestinal functionality of hybrid striped bass.

Fungal antioxidant pathways promote survival against neutrophils during infection

Filamentous fungi are a common cause of blindness and visual impairment worldwide. Using both murine model systems and in vitro human neutrophils, we found that NADPH oxidase produced by neutrophils was essential to control the growth of Aspergillus and Fusarium fungi in the cornea. We demonstrated that neutrophil oxidant production and antifungal activity are dependent on CD18, but not on the β-glucan receptor dectin-1. We used mutant A. fumigatus strains to show that the reactive oxygen species-sensing transcription factor Yap1, superoxide dismutases, and the Yap1-regulated thioredoxin antioxidant pathway are each required for protection against neutrophil-mediated oxidation of hyphae as well as optimal survival of fungal hyphae in vivo. We also demonstrated that thioredoxin inhibition using the anticancer drug PX-12 increased the sensitivity of fungal hyphae to both H2O2- and neutrophil-mediated killing in vitro. Additionally, topical application of PX-12 significantly enhanced neutrophil-mediated fungal killing in infected mouse corneas. Cumulatively, our data reveal critical host oxidative and fungal anti-oxidative mediators that regulate hyphal survival during infection. Further, these findings also indicate that targeting fungal anti-oxidative defenses via PX-12 may represent an efficacious strategy for treating fungal infections.

Evaluation of the Ability of GroBiotic®‐A to Enhance Growth, Muscle Composition, Immune Responses, and Resistance Against Aeromonas hydrophila in Nile tilapia, Oreochromis niloticus

This study was conducted to evaluate the potential of graded levels of GroBiotic®‐A to improve performance of Nile tilapia, Oreochromis niloticus, fed a 29% crude protein (CP) diet. A 29% CP diet was formulated and supplemented with 0, 0.4, 0.8, and 1.2% GroBiotic®‐A and compared to performance of fish fed a 33% CP diet. Enhanced weight gain and feed efficiency were generally observed in fish fed the diets supplemented with GroBiotic®‐A compared to the 29% CP diet. No significant differences in these responses were observed between fish fed diets supplemented with GroBiotic®‐A compared to those fed the 33% CP diet. Supplementation of 0.8 and 1.2% GroBiotic®‐A induced significantly lower condition factor and hepatosomatic index compared to fish fed the 29% CP diet, but those values were similar to that of fish fed the 33% CP diet. GroBiotic®‐A supplementation and protein reduction had no effect on the viscerosomatic index of fish or moisture, lipid, and protein content of muscle samples. However, muscle ash increased significantly with protein reduction (29% CP diet), but GroBiotic®‐A supplementation (0.8 and 1.2%) reduced muscle ash content. Activities of catalase and superoxide dismutase were markedly reduced in fish fed GroBiotic®‐A (0.8 and 1.2%) compared to those fed the control diet. GroBiotic®‐A supplementation also induced significantly higher neutrophil oxidative radical production compared to fish fed the 29% CP diet, but no significant difference was observed in comparison with the 33% CP diet. After 8 wk of feeding, exposure to Aeromonas hydrophila for 3 wk resulted in 40% (0.4, 0.8% GroBiotic®‐A) and 27% (1.2% GroBiotic®‐A) mortality and reduced signs of disease, while 47% mortality was observed in fish fed the 29% CP diet. Based on the result of this study, it is concluded that 0.8 and 1.2% GroBiotic®‐A positively influenced growth performance and feed efficiency of tilapia fed diets containing 29% crude protein to levels comparable to fish fed the 33% CP diet. GroBiotic®‐A supplementation also significantly increased neutrophil oxidative radical production as well as resistance to Ae. hydrophila infection.

Effects of dietary arginine and glutamine on growth performance, immune responses and intestinal structure of red drum, Sciaenops ocellatus

Both arginine and glutamine play important roles in tissue repair, cell replication and collagen synthesis which impact animal growth and survival. Thus, a 7-week feeding trial was conducted to determine the effects of dietary arginine and glutamine on growth performance, innate immune responses and intestinal structure of juvenile red drum, Sciaenops ocellatus (6.9 ± 0.04 g, initial average weight). Protein in all experimental diets was derived from menhaden fishmeal and dehulled, solvent-extracted soybean meal to provide isonitrogenous (44% crude protein) and isolipidic (10%) diets. The basal diet contained 0.14% glutamine and 1.72% arginine on a dry-matter basis, which exceeds the established dietary requirement of red drum. Experimental diets were prepared by supplementing the basal diet with either arginine at 1% of dry weight, glutamine at 1%, glutamine at 2%, or both arginine and glutamine at 1%, with adjustments in glycine to maintain equal nitrogen among all diets. Feed efficiency was significantly ( P < 0.05) improved by supplementation of glutamine at 2% and the combination of both arginine and glutamine at 1% of diet. Neutrophil oxidative radical production in fish fed the glutamine and/or arginine-supplemented diets was significantly ( P = 0.03) higher compared with that of fish fed the basal diet, with a synergistic effect observed in fish fed the combined arginine–glutamine diet. Significantly ( P < 0.05) higher serum lysozyme activity also was observed in fish fed the diet supplemented with 1% of both arginine and glutamine. Extracellular superoxide anion production by red drum macrophages was significantly ( P < 0.05) higher for fish fed diets with glutamine at either 1 or 2% and the 1% arginine–glutamine combination compared to fish fed the basal diet. Similarly, macrophages from fish fed the diet with glutamine at 2% and the arginine–glutamine combination at 1% produced significantly higher amounts of intracellular superoxide anion. Morphometric analyses – i.e., measurements of enterocyte, microvillus and fold heights – demonstrated positive effects of both dietary glutamine and arginine in different portions (proximal, mid and distal) of the gastrointestinal tract of red drum. In general, fish fed the diet supplemented with 2% glutamine had the greatest increases in magnitude of the chosen structures, although those fed the diet with arginine at 1% also yielded improved scores for some enteric portions. Therefore, results from the present study establish the importance of both dietary arginine and glutamine supplementation in improving feed efficiency, as well as eliciting positive changes to several components of the innate immune system and intestinal functionality of red drum.

Extracellular ATP induces spikes in cytosolic free Ca2+ but not in NADPH oxidase activity in neutrophils

In order to establish whether non-mitochondrial oxidase activity in human neutrophils is tightly related to cytosolic Ca2+ concentration, we simultaneously measured Ca2+ oscillations induced by ATP and oxidant production in single adherent neutrophils using confocal microscopy. ATP induced fast damped Ca2+ spikes with a period of 15s and slower irregular spikes with a period greater than 50s. Spikes in Ca2+ occurred in the absence of Ca2+ influx, but the amplitude was damped by inhibition of Ca2+ influx. Using the oxidation of hydroethidine as a cytosolic marker of oxidant production, we show that the generation of reactive oxygen species by neutrophils adherent to glass was accelerated by ATP. The step-up in NADPH oxidase activity followed the first elevation of cytosolic Ca2+ but, despite subsequent spikes in Ca2+ concentration, no oscillations in oxidase activity could be detected. ATP induced spikes in Ca2+ in a very reproducible way and we propose that the Ca2+ signal is an on-switch for oxidase activity, but the activity is apparently not directly correlated with spiking activity in cytosolic Ca2+.

Effects of dietary prebiotics on growth performance, immune response and intestinal morphology of red drum ( Sciaenops ocellatus)

An 8-week feeding trial was conducted with juvenile red drum to evaluate four different prebiotics: fructooligosaccharides (FOS) in the form of inulin, galactooligosaccharides (GOS), Bio-MOS ®, containing mannanoligosaccharides (MOS) derived from yeast, and Previda™ containing galacto-gluco-mannans from hemicellulose extract. Each prebiotic was added at a level of 10 g kg −1 to the basal diet which was formulated to contain 41% protein and 10% lipid. Each diet was randomly assigned to three aquaria and fed twice daily to apparent satiation. Fish fed the diet containing Previda™ had significantly higher ( P < 0.05) weight gain than fish fed the basal diet and the one supplemented with Bio-MOS ®. Feed efficiency and protein efficiency ratio of fish fed the various diets were not significantly different ( P > 0.05), although fish fed the basal diet had the lowest values. Fish fed the Bio-MOS ® diet had lower survival than fish fed the other diets. Hepatosomatic index, muscle ratio and condition factor were not affected by the dietary treatments. Fish fed the FOS diet had a significantly lower neutrophil oxidative radical production than fish fed the other diets. However, serum lysozyme activity was significantly lower ( P < 0.05) in fish fed the basal diet compared with those fed the diets supplemented with prebiotics. Quantitative changes in the ultrastructural characteristics of the gastrointestinal tract of red drum fed the various diets were evaluated using histological methods. Fold height and enterocyte height in pyloric caeca and proximal, mid- and distal intestines were not significantly affected by diet. However, microvilli heights in pyloric caeca, proximal and mid-intestine were significantly increased by the supplementation with prebiotics. In this experiment, Previda™ supplementation resulted in significant improvements in growth performance and immunological responses, even as compared to other commercially available prebiotics. Because prebiotic supplementation may result in enhancement of different performance indicators in various fish species, tailored evaluations are recommended before suggesting specific prebiotic strategies.

A Novel Function of Sphingosine Kinase 1 Suppression of JNK Activity in Preventing Inflammation and Injury

The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappaB activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.

N-Acetylcysteine Inhibits RhoA and Promotes Apoptotic Cell Clearance during Intense Lung Inflammation

The resolution of pulmonary inflammation seen in various inflammatory lung conditions depends on the clearance of apoptotic cells to prevent permanent tissue damage or progressive disease. Uptake of apoptotic cells by alveolar macrophages is suppressed by oxidants through the activation of Rho signaling. We hypothesized that antioxidant exposure would increase the ability of alveolar macrophages to clear pulmonary apoptotic cells through the inhibition of RhoA. The effects of the antioxidant N-acetylcysteine (NAC) on the pulmonary immune response were seen in mice treated intratracheally with LPS, LPS + NAC, or saline. Apoptotic cell clearance, RhoA activity, and changes in the lung inflammatory responses were analyzed in vivo or ex vivo. Neutrophil accumulation, apoptosis, necrosis, and oxidant production peaked at 3 days post LPS treatment. NAC enhanced the clearance of apoptotic cells and inhibited RhoA activity in alveolar macrophages at 3 days post LPS treatment. NAC suppressed LPS-induced proinflammatory mediators, enhanced the production of transforming growth factor-beta1, reduced the accumulation of inflammatory cells, and reduced levels of protein and lactate dehydrogenase in bronchoalveolar lavage fluid. In the presence of ex vivo apoptotic cells, alveolar macrophages exposed to LPS or LPS + NAC had reduced tumor necrosis factor-alpha levels and increased transforming growth factor-beta1 levels. A Rho kinase inhibitor mimicked the effects of NAC on the clearance of apoptotic cells and the inflammatory responses. These results indicate that NAC can expedite the resolution of LPS-induced pulmonary inflammation through the inhibition of RhoA activity and the enhancement of apoptotic cell clearance.

A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity

AbstractChronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91phox and p22phox, which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47phox and p67phox. A fifth subunit, p40phox, plays an important role in phagocytosis-induced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40phox, in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40phoxR105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40phox-deficient granulocytes, with premature loss of p40phoxR105Q from phagosomes. Thus, p40phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease.