Phagocytic Reactive Oxygen Species Research Articles

Effect of proteins from the coelomic fluid of superficially wounded holothurians on the activity of phagocytes

Holothurians are among the most capable of regenerating animals. Their phagocytes are analogues of macrophages, but the mechanisms of their participation in wound healing have not been studied. The aim of the work is to determine the effect of individual protein components isolated from the coelomic fluid of holothurians with superficial damage to the body wall on the functional activity of the two types of phagocytes (P1 and P2) in holothurian Eupentacta fraudatrix. Protein components of the coelomic fluid of holothurians with superficial wounds of the body wall were analyzed and collected by gel permeation chromatography. We used proteins whose content changed significantly during the wound healing period in preliminary experiments. Two proteins and a peptide were added to phagocytes, isolated by gradient centrifugation, simultaneously with the thermostable toxin of Yersinia pseudotuberculosis and incubated for 24 h. The production of superoxide anion radical was determined by the reduction of nitroblue tetrazolium (NBT) using a colorimetric method. To assess the specificity of the proteins, bovine serum albumin (BSA) was used as an internal control. In one-day in vivo experiments, proteins were administered to wounded animals. Two proteins, when compared with the effect of BSA, did not show a specific effect on the production of reactive oxygen species in phagocytes in vitro. However, a comparison of the effect of the peptide on the oxidative activity and viability of phagocytes with those of BSA revealed the specificity of its action. The peptide reduced the oxidative activity of P1 phagocytes and increased it in P2 phagocytes in a direct concentration-dependent manner. One of the two proteins being administered to wounded holothurians caused a multidirectional concentration-dependent effect on the oxidative activity of the two types of phagocytes, with a predominant suppression of the P1 phenotype activity. The ability of protein components to cause a shift in the ratio of oxidative activity of the two types of phagocytes towards the preferential activation of P2 phagocytes, which are considered as functional analogues of M2 macrophages, suggests that they act as anti-inflammatory agents.

Detoxification of reactive oxygen species by the hyaluronic acid capsule of group A Streptococcus.

The pro-inflammatory cytokine IL-6 regulates antimicrobial responses that are broadly crucial in the defense against infection. Our prior work shows that IL-6 promotes the killing of the M4 serotype group A Streptococcus (GAS) but does not impact the globally disseminated M1T1 serotype associated with invasive infections. Using in vitro and in vivo infection models, we show that IL-6 induces phagocyte reactive oxygen species (ROS) that are responsible for the differential susceptibility of M4 and M1T1 GAS to IL-6-mediated defenses. Clinical isolates naturally deficient in capsule, or M1T1 strains deficient in capsule production, are sensitive to this ROS killing. The GAS capsule is made of hyaluronic acid, an antioxidant that detoxifies ROS and can protect acapsular M4 GAS when added exogenously. During in vitro interactions with macrophages and neutrophils, acapsular GAS can also be rescued with the antioxidant N-acetylcysteine, suggesting this is a major virulence contribution of the capsule. In an intradermal infection model with gp91phox -/- (chronic granulomatous disease [CGD]) mice, phagocyte ROS production had a modest effect on bacterial proliferation and the cytokine response but significantly limited the size of the bacterial lesion in the skin. These data suggest that the capsule broadly provides enhanced resistance to phagocyte ROS but is not essential for invasive infection. Since capsule-deficient strains are observed across several GAS serotypes and are competent for transmission and both mild and invasive infections, additional host or microbe factors may contribute to ROS detoxification during GAS infections.

Specification of hemocyte subpopulations based on immune-related activities and the production of the agglutinin MkC1qDC in the bivalve Modiolus kurilensis

Bivalves, such as Modiolus are used as indicator organisms to monitor the state of the marine environment. Even though hemocytes are known to play a key role in the adaptive and protective mechanisms of bivalves, these cells are poorly studied in horse-mussel Modiolus kurilensis. In this paper, we present classification of horse-mussel hemocytes based on their immune functions, including the production of specific immune-related molecules, as well as their morphological composition after isolation by density gradient centrifugation. An effective fractionation protocol was adapted to separate four hemocyte subpopulations with distinct morphofunctional profiles. First subpopulation consisted of small under-differentiated hemoblasts (2.20 ± 0.85%) with a bromodeoxyuridine positive nucleus, and did not show any immune reactivity. Second was represented by agranulocytes (24.11 ± 2.40%), with evenly filled cytoplasm containing a well-developed protein-synthesizing apparatus, polysomes, smooth endoplasmic reticulum and mitochondria, and positively stained for myeloperoxidase, acidic proteins, glycogen and neutral polysaccharides. Third subpopulation consisted of eosinophilic granulocytes (62.64 ± 9.32%) that contained the largest number of lysosomes, peroxisomes and vesicles with contents of different density, and showed the highest phosphatase, reactive oxygen species (ROS) and phagocytic activities. Lastly, fourth group, basophilic granulocytes (14.21 ± 0.34%), are main producers of lectin-like protein MkC1qDC, recently discovered in M. kurilensis and characterized by pronounced antibacterial and anticancer activity. These cells characterized by intracytoplasmic of the MkC1qDC localization, forming granule-like bodies visualized with specific antibody. Both granulocytes and agranulocytes showed phagocytic activity and ROS production, and these reactions were more pronounced for eosinophilic granulocytes, suggesting that this group is the key element of the cell-mediated immune response of M. kurilensis. Our results support a concept of bivalve's hemocyte specification with distinct phenotypes.

The use of routine dihydrorhodamine-123 testing in suspected cases of primary immune deficiency

Chronic granulomatous disease (CGD) is a rare inborn error of immunity occurring in approximately 1:200 000-250 000 live births. It is characterised by the defective formation of reactive oxygen species in phagocytes due to mutations in the genes encoding the proteins that form the NADPH oxidase complex. Infants are born asymptomatic but have a susceptibility to infections, granulomatous inflammation, and immune dysregulation. Curative therapy is available in the form of haematopoietic stem cell transplant, and gene therapy is also emerging as an alternative curative option.

Gastrointestinal and Hepatic Manifestations of Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) is a rare inborn error of immunity, resulting from a defect in nicotinamide adenine dinucleotide phosphate oxidation and decreased production of phagocyte reactive oxygen species. The main clinical manifestations are recurrent infections and chronic inflammatory disorders. Current approaches to management include antimicrobial prophylaxis and control of inflammatory complications. Hematopoietic stem cell transplantation or gene therapy can provide definitive treatment. Gastrointestinal and hepatic manifestations are common in CGD and include structural changes, dysmotility, CGD-associated inflammatory bowel disease, liver abscesses, and noncirrhotic portal hypertension. The findings can be heterogeneous, and the management is complex in light of the underlying immune dysfunction. This review describes the various clinical findings and the latest studies in management of gastrointestinal and hepatic manifestations in CGD, as well as the managementexperience at the National Institutes of Health.

IMMUNOMODULATORY POTENTIAL OF CLAUSENA EXCAVATA LEAVES FRACTIONS VIA DECREASING THE PRODUCTION OF REACTIVE OXYGEN SPECIES FROM IMMUNE CELLS

Objective: Clausena excavata is known to possess anti-oxidant property. However, this property through which mechanism it affects the immune cells and suppresses the production of reactive oxygen species (ROS) has not been explored. Material and Method: This study evaluated the immunomodulatory activities of ethyl acetate, petroleum ether, chloroform, and methanol C. excavata leaf extracts by decreasing the production of ROS from whole blood, polymorphonuclears (PMNs) cells and macrophages. Result and Discussion: Among the fractions tested, ethyl acetate C. excavate extract (EACE) showed potent anti-oxidant property and significantly (p < 0.001) suppressed intracellular and extracellular phagocytic oxidative ROS burst produced by the zymosan and PMA-activated whole blood, PMNs, and macrophages cells with 50% inhibitory concentration (IC50) values of 5.7 ± 0.01, 1.3 ± 0.01, and 0.7 ± 0.03 µg/mL respectively. This study provides information regarding the mechanism behind its anti-oxidant property and its herbal use in treating various higher oxidative stress associated diseases.

Prostate cancer addiction to oxidative stress defines sensitivity to anti-tumor neutrophils

Bone metastatic prostate cancer (BM-PCa) remains one of the most difficult cancers to treat due to the complex interactions of cancer and stromal cells. We previously showed that bone marrow neutrophils elicit an anti-tumor immune response against BM-PCa. Further, we demonstrated that BM-PCa induces neutrophil oxidative burst, which has previously been identified to promote primary tumor growth of other cancers, and a goal of this study was to define the importance of neutrophil oxidative burst in BM-PCa. To do this, we first examined the impact of depletion of reactive oxygen species (ROS), via systemic deletion of the main source of ROS in phagocytes, NADPH oxidase (Nox)2, which we found to suppress prostate tumor growth in bone. Further, using pharmacologic ROS inhibitors and Nox2-null neutrophils, we found that ROS depletion specifically suppresses growth of androgen-insensitive prostate cancer cells. Upon closer examination using bulk RNA sequencing analysis, we identified that metastatic prostate cancer induces neutrophil transcriptomic changes that activates pathways associated with response to oxidative stress. In tandem, prostate cancer cells resist neutrophil anti-tumor response via extracellular (i.e., regulation of neutrophils) and intracellular alterations of glutathione synthesis, the most potent cellular antioxidant. These findings demonstrate that BM-PCa thrive under oxidative stress conditions and such that regulation of ROS and glutathione programming could be leveraged for targeting of BM-PCa progression.

Low density neutrophils are increased in patients with Beh\xe7et\u2019s disease but do not explain differences in neutrophil function

BackgroundBehçet’s disease (BD) is a multisystem autoinflammatory disease characterised by mucosal ulceration, ocular, neural, joint and skin inflammation. The cause of BD is not known but there is a strong genetic association with HLA-B*51, IL10 and IL23R. Neutrophils are a first line of defence against invading pathogens and have been described as activated in patients with BD. Neutrophils can now be separated into different subsets, such as low density (LDN) and normal density (NDN) that have diverse functional roles. We wished to address neutrophil heterogeneity in patients with BD.MethodsPeripheral blood neutrophils were obtained from 32 BD patients and 37 healthy aged-matched controls. Percoll isolation was used to isolate all neutrophils, while Ficol-Hypaque was used to obtain LDN and NDN. Phagocytic capacity and production of reactive oxygen species (ROS), and neutrophil extracellular traps (NET) stimulated with phorbol 12-myristate 13-acetate (PMA) and Escherichia coli (E.coli) were assessed in both groups.ResultsWe have demonstrated reduced phagocytic capacity and ROS production but greater NET production by total neutrophils stimulated with PMA or E.coli from BD patients in comparison with healthy controls. Patients with BD had elevated numbers of LDN and lower number of NDN compared with healthy controls. However, both neutrophil subsets showed the same reduced ROS production and phagocytic function as total neutrophils in both groups.ConclusionOur novel findings indicate that the neutrophil population in BD is heterogeneous and the increased number of LDN in combination with greater NET production may contribute to the inflammatory response and pathogenesis.

Ferumoxytol-β-glucan Inhibits Melanoma Growth via Interacting with Dectin-1 to Polarize Macrophages into M1 Phenotype.

Background: Regulating the polarization of macrophages to antitumor M1 macrophages is a promising strategy for overcoming the immunosuppression of the tumor microenvironment for cancer therapy. Ferumoxytol (FMT) can not only serve as a drug deliver agent but also exerts anti-tumor activity. β-glucan has immuno-modulating properties to prevent tumor growth. Thus, a nanocomposite of FMT surface-coated with β-glucan (FMT-β-glucan) was prepared to explore its effect on tumor suppression.Methods: Male B16F10 melanoma mouse model was established to explore the antitumor effect of FMT-β-glucan. The viability and apoptotic rates of B16F10 cells were detected by cell counting kit-8 and Annexin-V/PI experiments. The levels of M1 markers were quantified by quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay. Phagocytic activity and intracellular reactive oxygen species (ROS) in macrophages were evaluated by the neutral red uptake assay and flow cytometry, respectively. Small interfering RNA (siRNA) transfection was applied to knock down the Dectin-1 gene in RAW 264.7 cells.Results: FMT-β-glucan suppressed tumor growth to a greater extent and induced higher infiltration of M1 macrophages than the combination of FMT and β-glucan (FMT+β-glucan) in vivo. In vitro, supernatant from FMT-β-glucan-treated RAW 264.7 cells led to lower cell viability and induced more apoptosis of B16F10 cells than that from the FMT+β-glucan group. Moreover, FMT-β-glucan boosted the expression of M1 type markers, and increased phagocytic activity and ROS in RAW 264.7 cells. Further research indicated that FMT-β-glucan treatment promoted the level of Dectin-1 on the surface of RAW 264.7 cells and that knockdown of Dectin-1 abrogated the phosphorylation levels of several components in MAPK and NF-κB signaling.Conclusion: The nanocomposite FMT-β-glucan suppressed melanoma growth by inducing the M1 macrophage-activated tumor microenvironment.

LETMD1 Regulates Phagocytosis and Inflammatory Responses to Lipopolysaccharide via Reactive Oxygen Species Generation and NF-κB Activation in Macrophages.

LETM1 domain-containing protein 1 (LETMD1), also known as HCCR-1, is a mitochondrial protein and is known to regulate p53 and STAT3 activities in cancer cells. In this study, we present, for the first time (to our knowledge), data indicating that LETMD1 suppresses multiple immune responses in monocyte/macrophage lineage cells and mouse primary macrophages. Attenuation of LETMD1 expression with specific small interfering RNA and short hairpin RNA constructs enhanced LPS-induced expressions of inflammatory mediators in macrophages. In addition, LETMD1 attenuation caused potentiation of phagocytosis as well as migration in a macrophage-like cell line, U937. These enhancing effects were associated with altered activation of signaling adaptors (such as NF-κB, MAPKs, p53, and JAK-STAT) involved in TLR4 signaling. Especially, LETMD1 selectively regulated TLR4-induced NF-κB activation via MyD88 but not via TIR-domain-containing adapter-inducing IFN-β (TRIF). Attenuation of LETMD1 expression caused mitochondrial hyperpolarization and subsequent decrease in ATP production and increase in mitochondrial/cellular reactive oxygen species (ROS) and intracellular calcium levels. LETMD1 attenuation also enhanced LPS-induced expression of NADPH oxidase (NOX) 2, the main producer of cellular ROS in phagocytes, through augmenting IFN regulatory factor 1. Accordingly, treatment with ROS scavenger, NOX2 suppressing agents, or calcium chelators resulted in suppression of LPS-induced cytokine production as well as NF-κB activation in cells with LETMD1 attenuation. These findings reveal a previously unknown function of LETMD1 and provide evidences showing LETMD1 negatively regulates macrophage functions by modulating mitochondrial function, subsequent ROS generation, and NF-κB activation.

Vitex negundo L. leaf extract inhibits IL-6 and TNF-α secretion and phagocytosis in human leukocytes

Vitex negundo L. is used as an anti-inflammatory agent in Ayurveda and Sri Lankan traditional medicine. Here, the anti-inflammatory effects of an aqueous leaf extract (ALE) of V. negundo L. was investigated. The inhibitory effects of freeze-dried ALE (15.7–500 μg/mL) against interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumour necrosis factor-α (TNFα) secretion by human mononuclear cells, phagocytic activity of human neutrophils, and reactive oxygen species (ROS) and nitric oxide (NO) production by human leukocytes were tested in vitro. The inhibitory effects were dose dependent, and the strongest effect was observed at 500 μg/mL (P < 0.05). ALE caused 58.0 ± 1.0 % and 26.1 ± 2.0 % inhibition of IL-6 and TNFα secretion, respectively, whereas the positive control, Andrographis paniculata, caused complete inhibition. Phagocytic activity and ROS production were inhibited by 55 ± 4.5 % and 58.6 ± 8.6 %, respectively, whereas the positive controls, Ixora coccinea and diphenyleneiodonium chloride, caused 100 ± 4.4 % and 80 ± 6.4 % inhibition, respectively. ALE markedly inhibited NO production (87.5 ± 17.7 %), and the effect was comparable to that of the positive control, N-monomethyl-l-arginine (95.0 ± 1.0 %). The membrane-stabilizing activity of ALE (0.01–100 μg/mL) was assessed using heat-induced lysis of human erythrocytes. The highest membrane stabilizing activity (91 %) was observed at 0.01 μg/mL and was comparable with that of aspirin (positive control; 86.6 ± 0.51 %). These inhibitory effects may be the mechanisms through which V. negundo L. elicits its anti-inflammatory activities and support the traditional use of V. negundo L. as an anti-inflammatory agent.

Decreased integrity of exercise-induced plasma cell free nuclear DNA \u2013 negative association with the increased oxidants production by circulating phagocytes

Strenuous exercise increases circulating cell free DNA (cfDNA) and stimulates blood phagocytes to generate reactive oxygen species (ROS) which may induce DNA strand breaks. We tested whether: (A) elevated cfDNA in response to three repeated bouts of exhaustive exercise has decreased integrity; (B) each bout of exercise increases luminol enhanced whole blood chemiluminescence (LBCL) as a measure of ROS production by polymorphonuclear leukocytes. Eleven men performed three treadmill exercise tests to exhaustion separated by 72 hours of resting. Pre- and post-exercise concentrations and integrity of cf nuclear and mitochondrial DNA (cf n-DNA, cf mt-DNA) and resting (r) and fMLP (n-formyl-methionyl-leucyl-phenylalanine)-stimulated LBCL were determined. Each bout increased concentrations of cf n-DNA by more than 10-times which was accompanied by about 2-times elevated post-exercise rLBCL and fMLP-LBCL. Post-exercise cf n-DNA integrity (integrity index, I229/97) decreased after the first (0.59 ± 0.19 vs. 0.48 ± 0.18) and second (0.53 ± 0.14 vs. 0.44 ± 0.17) bout of exercise. There were negative correlations between I229/97 and rLBCL (ƍ = –0.37), and I229/97 and fMLP-LBCL (ƍ = –0.40) – analysis of pooled pre- and post-exercise data (n = 66). cf mt- DNA integrity (I218/78) did not alter in response to exercise. This suggests an involvement of phagocyte ROS in cf n-DNA strand breaks in response to exhaustive exercise.

Major CD4 T-Cell Depletion and Immune Senescence in a Patient with Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) results from primary defects in phagocytic reactive oxygen species (ROS) production. T-cell evaluation is usually neglected during patients’ follow-up, although T-cell depletion has been reported in CGD through unknown mechanisms. We describe here a 36-year-old patient with X-linked CGD with severe CD4 T-cell depletion <200 CD4 T-cells/μl, providing insights into the mechanisms that underlie T-cell loss in the context of oxidative burst defects. In addition to the typical infections, the patient featured a progressive T-cell loss associated with persistent lymphocyte activation, expansion of interleukin (IL)-17-producing CD4 T-cells, and impaired thymic activity, leading to a reduced replenishment of the T-cell pool. A relative CD4 depletion was also found at the gut mucosal level, although no bias to IL-17-production was documented. This immunological pattern of exhaustion of immune resources favors prompt, potentially curative, therapeutic interventions in CGD patients, namely, stem-cell transplantation or gene therapy. Moreover, this clinical case raises new research questions on the interplay of ROS production and T-cell homeostasis and immune senescence.

Severe glucose-6-phosphate dehydrogenase deficiency leads to susceptibility to infection and absent NETosis

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymatic disorder of red blood cells in human subjects, causing hemolytic anemia linked to impaired nicotinamide adenine dinucleotide phosphate (NADPH) production and imbalanced redox homeostasis in erythrocytes. Because G6PD is expressed by a variety of hematologic and nonhematologic cells, a broader clinical phenotype could be postulated in G6PD-deficient patients. We describe 3 brothers with severe G6PD deficiency and susceptibility to bacterial infection. We sought to study the molecular pathophysiology leading to susceptibility to infection in 3 siblings with severe G6PD deficiency. Blood samples of 3 patients with severe G6PD deficiency were analyzed for G6PD enzyme activity, cellular oxidized nicotinamide adenine dinucleotide phosphate/NADPH levels, phagocytic reactive oxygen species production, neutrophil extracellular trap (NET) formation, and neutrophil elastase translocation. In these 3 brothers strongly reduced NADPH oxidase function was found in granulocytes, leading to impaired NET formation. Defective NET formation has thus far been only observed in patients with the NADPH oxidase deficiency chronic granulomatous disease, who require antibiotic and antimycotic prophylaxis to prevent life-threatening bacterial and fungal infections. Because severe G6PD deficiency can be a phenocopy of chronic granulomatous disease with regard to the cellular and clinical phenotype, careful evaluation of neutrophil function seems mandatory in these patients to decide on appropriate anti-infective preventive measures. Determining the level of G6PD enzyme activity should be followed by analysis of reactive oxygen species production and NET formation to decide on required antibiotic and antimycotic prophylaxis.

Saxitoxin Modulates Immunological Parameters and Gene Transcription in Mytilus chilensis Hemocytes.

Saxitoxin (STX) is a neurotoxin produced by dinoflagellates in diverse species, such as Alexandrium spp., and it causes paralytic shellfish poisoning (PSP) in humans after the ingestion of contaminated shellfish. Recent studies have suggested that the immune functions of bivalves could be affected by harmful algae and/or by their toxins. Herein, hemocytes are the main effector cells of the immune cellular response. In this study, we evaluated the response of hemocytes from the mussel Mytilus chilensis to STX exposure in a primary culture. Cell cultures were characterized according to size and complexity, while reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein diacetate (DCFH-DA) assay. Finally, phagocytic activity was measured using both flow cytometry and fluorescence microscopy assays. Additionally, gene transcription of candidate genes was evaluated by qPCR assays. The results evidenced that exposures to different concentrations of STX (1–100 nM) for 24 h did not affect cell viability, as determined by an MTT assay. However, when hemocytes were exposed for 4 or 16 h to STX (1–100 nM), there was a modulation of phagocytic activity and ROS production. Moreover, hemocytes exposed to 100 nM of STX for 4 or 16 h showed a significant increase in transcript levels of genes encoding for antioxidant enzymes (SOD, CAT), mitochondrial enzymes (COI, COIII, CYTB, ATP6, ND1) and ion channels (K+, Ca2+). Meanwhile, C-type lectin and toll-like receptor genes revealed a bi-phase transcriptional response after 16 and 24–48 h of exposure to STX. These results suggest that STX can negatively affect the immunocompetence of M. chilensis hemocytes, which were capable of responding to STX exposure in vitro by increasing the mRNA levels of antioxidant enzymes.

Synthesis and characterization of hetero-bimetallic complexes with 2-mercapto-5-methyl-benzimidazole: theoretical study and biological activities

Heterobimetallic complexes have been synthesized by stirring 2-mercapto-5-methylbenzimidazole with carbon disulfide in methanol at room temperature. In the second step, the product was treated with R2SnCl2/R3SnCl (R = Me, n-Bu, Ph) in 1 : 1 M ratio, then organotin(IV) complexes were treated with HgCl2 in 2 : 1 M : L ratio to yield heterobimetallic complexes. The ligand and complexes have been characterized by elemental analysis, IR, 1H- and 13C NMR spectroscopy, mass spectrometry (EI-MS), and semiempirical study to assess the binding mode of the heterobimetallic complexes. IR data showed the bidentate nature of the dithiocarbamate moiety, which is also confirmed by semiempirical study. Mass spectra correspond to the expected for the complexes. NMR spectroscopy confirmed the four-coordinate geometry in solution. Computed molecular descriptors, thermodynamic parameters, and electrostatic surface potential map of 4 were calculated by using the PM6 method. Biological screening data indicated that complexes exhibit significant activity against various bacterial and fungal strains with few exceptions. Ligand and 6 are excellent for phagocyte reactive oxygen species inhibition.

Chronic granulomatous disease presenting as refractory pneumonia in late adulthood

We present a case of refractory pneumonia in an adult patient with underlying chronic granulomatous disease (CGD). Her lobectomy tissue grew Burkholderia cepacia and histopathology revealed diffuse severe pneumonic consolidation with suppurative/necrotizing granulomata. An initial attempt to find an underlying immune deficiency was unsuccessful. Following recurrent invasive infections, repeat immunological assessment revealed reduced neutrophil function, demonstrating skewed carrier status (lyonization) for X-linked CGD (only 3% normal cells). A pathogenic mutation in the CYBB gene was found on sequencing. CYBB gene encodes the gp91phox, a catalytic subunit of nicotinamide adenine dinucleotide phosphate-oxidase that produces reactive oxygen species in phagocytes. Lyonization increases with age, explaining the delayed clinical CGD. CGD is a rare neutrophil disorder that usually presents in early life with recurrent infections due to bacteria and fungi primarily involving lungs and skin. It is secondary to a defective NADPH oxidase system needed to kill intracellular organisms and activate the formation of neutrophil extracellular traps.

Pioglitazone restores phagocyte mitochondrial oxidants and bactericidal capacity in chronic granulomatous disease

Deficient production of reactive oxygen species (ROS) by the phagocyte nicotinamide adenine dinucleotide (NADPH) oxidase in patients with chronic granulomatous disease (CGD) results in susceptibility to certain pathogens secondary to impaired oxidative killing and mobilization of other phagocyte defenses. Peroxisome proliferator-activated receptor (PPAR) γ agonists, including pioglitazone, approved for type 2 diabetes therapy alter cellular metabolism and can heighten ROS production. It was hypothesized that pioglitazone treatment of gp91(phox-/-) mice, a murine model of human CGD, would enhance phagocyte oxidant production and killing of Staphylococcus aureus, a significant pathogen in patients with this disorder. We sought to determine whether pioglitazone treatment of gp91(phox-/-) mice enhanced phagocyte oxidant production and host defense. Wild-type and gp91(phox-/-) mice were treated with the PPARγ agonist pioglitazone, and phagocyte ROS and killing of S aureus were investigated. As demonstrated by 3 different ROS-sensing probes, short-term treatment of gp91(phox-/-) mice with pioglitazone enhanced stimulated ROS production in neutrophils and monocytes from blood and neutrophils and inflammatory macrophages recruited to tissues. Mitochondria were identified as the source of ROS. Findings were replicated in human monocytes from patients with CGD after ex vivo pioglitazone treatment. Importantly, although mitochondrial (mt)ROS were deficient in gp91(phox-/-) phagocytes, their restoration with treatment significantly enabled killing of S aureus both ex vivo and in vivo. Together, the data support the hypothesis that signaling from the NADPH oxidase under normal circumstances governs phagocyte mtROS production and that such signaling is lacking in the absence of a functioning phagocyte oxidase. PPARγ agonism appears to bypass the need for the NADPH oxidase for enhanced mtROS production and partially restores host defense in CGD.

Reactive oxygen species deficiency induces autoimmunity with type 1 interferon signature.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in the phagocyte reactive oxygen species (ROS)-producing NOX2 enzyme complex and characterized by recurrent infections associated with hyperinflammatory and autoimmune manifestations. A translational, comparative analysis of CGD patients and the corresponding ROS-deficient Ncf1(m1J) mutated mouse model was performed to reveal the molecular pathways operating in NOX2 complex deficient inflammation. A prominent type I interferon (IFN) response signature that was accompanied by elevated autoantibody levels was identified in both mice and humans lacking functional NOX2 complex. To further underline the systemic lupus erythematosus (SLE)-related autoimmune process, we show that naïve Ncf1(m1J) mutated mice, similar to SLE patients, suffer from inflammatory kidney disease with IgG and C3 deposits in the glomeruli. Expression analysis of germ-free Ncf1(m1J) mutated mice reproduced the type I IFN signature, enabling us to conclude that the upregulated signaling pathway is of endogenous origin. Our findings link the previously unexplained connection between ROS deficiency and increased susceptibility to autoimmunity by the discovery that activation of IFN signaling is a major pathway downstream of a deficient NOX2 complex in both mice and humans. We conclude that the lack of phagocyte-derived oxidative burst is associated with spontaneous autoimmunity and linked with type I IFN signature in both mice and humans.

Protecting Against Post-influenza Bacterial Pneumonia by Increasing Phagocyte Recruitment and ROS Production

Seasonal and especially pandemic influenza predispose patients to secondary bacterial pneumonias, which are a major cause of deaths and morbidity. Staphylococcus aureus is a particularly common and deadly form of post-influenza pneumonia, and increasing staphylococcal drug resistance makes the development of new therapies urgent. We explored an innate immune-mediated model of the lung to define novel mechanisms by which the host can be protected against secondary staphylococcal pneumonia after sub-lethal influenza infection. We found that stimulating the innate immunity in the lung by overexpression of GM-CSF will result in resistance to S. aureus pneumonia after sublethal influenza infection. Resistance was mediated by alveolar macrophages and neutrophils, and was associated with increased production of reactive oxygen species (ROS) by alveolar macrophages. Resistance was abrogated by treatment with agents that scavenged ROS. We conclude that stimulating innate immunity in the lung markedly reduces susceptibility to post-influenza staphylococcal pneumonia and that this may represent a novel immunomodulatory strategy for prevention and treatment of secondary bacterial pneumonia after influenza.