We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for ...treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.
Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H2O2) production. Understanding the significance of this ...response is limited by the fact that the source of EGF-induced H2O2 production is unknown. Here we show that EGF-induced H2O2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1). Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H2O2 production in both cell lines. We also demonstrate that H2O2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1.
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•The Duox1-DuoxA1 complex is the predominant NADPH-oxidase in HaCaT and A431 cells of epidermal origin.•Epidermal growth factor (EGF) stimulates H2O2 production in a calcium-dependent way.•Downregulation of Duox1 or DuoxA1 expression abolishes the EGF-evoked H2O2 production.•Other mediators (niacin, ATPγS, TRPV4-agonist) are also able to activate Duox1 in epidermal cells.•Duox1 produced H2O2 leads to the oxidation of thioredoxin-1 and cytosolic peroxiredoxins.
Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory ...nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1). This enzyme can be activated by increasing cytosolic calcium levels. Using DUOX1 knockout animals as a model system we found an increased sensitivity towards certain noxious stimuli in DUOX1-deficient animals, which is not due to structural changes in the skin as evidenced by detailed immunohistochemical and electron-microscopic analysis of epidermal tissue. We show that DUOX1 is expressed in keratinocytes but not in the neural sensory pathway. The release of hydrogen peroxide by activated DUOX1 alters both the activity of neuronal TRPA1 and redox-sensitive potassium channels expressed in dorsal root ganglia primary sensory neurons. We describe hydrogen peroxide, produced by DUOX1 as a paracrine mediator of nociceptive signal transmission.
Our results indicate that a novel, hitherto unknown redox mechanism modulates noxious sensory signals.
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•DUOX1 is the most abundantly expressed NADPH oxidase in mouse keratinocytes.•Biotinyl tyramide-based method was devised to visualize DUOX1 produced H2O2in vitro.•DUOX1 knockout mice displayed enhanced thermal allodynic response.•H2O2 pretreated TRPA1 expressing cells reduced their responsiveness to TRPA1 agonists.•H2O2 modulates the redox state of Kcnq4 (Kv7.4) potassium channel.
Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of ...which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels.
The aim of this work was to study the dityrosine-forming activity of lactoperoxidase (LPO) and its potential application for measuring hydrogen peroxide (H2O2). It was observed that LPO was able to ...form dityrosine at low H2O2 concentrations. Since dityrosine concentration could be measured in a simple fluorimetric reaction, this activity of the enzyme was utilized for the measurement of H2O2 production in different systems. These experiments successfully measured the activity of NADPH oxidase 4 (Nox4) by this method. It was concluded that LPO-mediated dityrosine formation offers a simple way for H2O2 measurement.
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Dostopno za:
BFBNIB, DOBA, GIS, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We report the case of a 1-week-old male born full-term, who had two inconclusive severe combined immunodeficiency (SCID) newborn screens and developed scalp cellulitis and
bacteremia. He did not pass ...early confirmatory hearing screens. Initial blood counts and lymphocyte flow cytometry revealed profound neutropenia and lymphopenia with a T-/B-/NK- phenotype. Red blood cell adenosine deaminase 1 activity was within normal limits. A presumptive diagnosis of reticular dysgenesis was considered. Granulocyte colony-stimulating factor was started, but there was no improvement in neutrophil counts. Subsequent lymphocyte flow cytometry at around 4 weeks of age demonstrated an increase in T-, B- and NK-cell numbers, eliminating suspicion for SCID and raising concern for congenital neutropenia and bone marrow failure syndromes. Genetic testing revealed a novel variant in
c.181C>A (p.Gln61Lys) (Q61K). RAC2, a Ras-related GTPase, is the dominant RAC protein expressed in hematopoietic cells and is involved with various downstream immune-mediated responses. Pathogenic
variants show significant phenotypic heterogeneity (spanning from neutrophil defects to combined immunodeficiency) across dominant, constitutively activating, dominant activating, dominant negative, and autosomal recessive subtypes. Given the identification of a novel variant, functional testing was pursued to evaluate aberrant pathways described in other
pathogenic variants. In comparison to wild-type RAC2, the Q61K variant supported elevated superoxide production under both basal and PMA-stimulated conditions, increased PAK1 binding, and enhanced plasma membrane ruffling, consistent with other dominant, constitutively active mutations. This case highlights the diagnostic challenge associated with genetic variants identified via next-generation sequencing panels and the importance of functional assays to confirm variant pathogenicity.
Engagement of the BCR with Ags triggers signaling pathways for commitment of B lymphocyte responses that can be regulated, in part, by reactive oxygen species. To investigate the functional relevance ...of reactive oxygen species produced in primary B cells, we focused on the role of the hydrogen peroxide generator Duox1 in stimulated splenic B cells under the influence of the T
2 cytokine IL-4. We found that H
O
production in wild type (WT) and Nox2-deficient CD19
B cells was boosted concomitantly with enhanced expression of Duox1 following costimulation with BCR agonists together with IL-4, whereas stimulated Duox1
cells showed attenuated H
O
release. We examined whether Duox1-derived H
O
contributes to proliferative activity and Ig isotype production in CD19
cells upon BCR stimulation. Duox1
CD19
B cells showed normal responses of Ig production but a higher rate of proliferation than WT or Nox2-deficient cells. Furthermore, we demonstrated that the H
O
scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of WT CD19
B cells in vitro. Results from immunized mice reflected the in vitro observations: T cell-independent Ag induced increased B cell expansion in germinal centers from Duox1
mice relative to WT and Nox2
mice, whereas immunization with T cell-dependent or -independent Ag elicited normal Ig isotype secretion in the Duox1 mutant mice. These observations, obtained both by in vitro and in vivo approaches, strongly suggest that Duox1-derived hydrogen peroxide negatively regulates proliferative activity but not Ig isotype production in primary splenic CD19
B cells.
In the thyroid gland Duox2-derived H2O2 is essential for thyroid hormone biosynthesis. Several patients were identified with partial or severe iodide organification defects caused by mutation in the ...gene for Duox2 or its maturation factor, DuoxA2. A Duox2-deficient (Duox2thyd) mouse model enabled in vivo investigation of its critical function in thyroid tissues, but its roles proposed in host defense or other innate responses in nonthyroid tissues remain less certain. These mice carry a spontaneous DUOX2 missense mutation, a T→G transversion, in exon 16 that changes the highly conserved valine 674 to glycine and results in severe congenital hypothyroidism. The exact mechanism underlying the effects of the V674G mutation has not been elucidated at the molecular or cellular level. To determine how the V674G mutation leads to congenital hypothyroidism, we introduced the same mutation into human Duox2 or Duox1 cDNAs and expressed them in HEK-293 cells stably expressing the corresponding DuoxA proteins. We found that the valine→glycine mutant Duox proteins fail to produce H2O2, lose their plasma membrane localization pattern, and are retained within the endoplasmic reticulum. The Duox2 mutant binds to DuoxA2, but appears to be unstable owing to this retention. Immunohistochemical staining of Duox2 in murine salivary gland ducts showed that Duox2 in mutant mice loses its condensed apical plasma membrane localization pattern characteristic of wild-type Duox2 and accumulates in punctate vesicular structures within cells. Our findings demonstrate that changing the highly conserved valine 674 in Duox2 leads to impaired subcellular targeting and reactive oxygen species release required for hormonogenesis, resulting in congenital hypothyroidism.
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•We explored the effects of the Duox2thyd mutation causing hypothyroidism in mice.•V674G Duox2 and V670G Duox1 failed to produce extracellular hydrogen peroxide.•Mutant Duox forms a complex with its maturation factor but is retained in the ER.•Staining of salivary glands from thyd mice verified the Duox2 translocation defect.
The p22phox protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22phox in the ...TGF-β1-stimulated H2O2 production of primary human and murine fibroblasts. TGF-β1 induced H2O2 release of the examined cells, and the response was dependent on the expression of both Nox4 and p22phox. Interestingly, the p22phox protein was present in the absence of any detectable Nox/Duox expression, and the p22phox level was unaffected by TGF-β1. On the other hand, Nox4 expression was dependent on the presence of p22phox, establishing an asymmetrical relationship between the two proteins. Nox4 and p22phox proteins localized to the endoplasmic reticulum and their distribution was unaffected by TGF-β1. We used a chemically induced protein dimerization method to study the orientation of p22phox and Nox4 in the endoplasmic reticulum membrane. This technique is based on the rapamycin-mediated heterodimerization of the mammalian FRB domain with the FK506 binding protein. The results of these experiments suggest that the enzyme complex produces H2O2 into the lumen of the endoplasmic reticulum, indicating that Nox4 contributes to the development of the oxidative milieu within this organelle.
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•The Nox4 protein expression is regulated by the presence of p22phox in primary fibroblasts.•The p22phox protein is stable in the absence of detectable Nox4 expression.•Nox4 and p22phox co-localize in the endoplasmic reticulum.•Orientation of the enzyme complex suggests H2O2 production into the ER lumen.
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