Effects of IFN-γ on intracellular trafficking and activity of macrophage NADPH oxidase flavocytochrome b558

ABSTRACT Phagocytic cells are the first line of innate defense against intracellular pathogens, and yet Toxoplasma gondii is renowned for its ability to survive in macrophages, although this paradigm is based on virulent type I parasites. Surprisingly, we find that avirulent type III parasites are preferentially cleared in naive macrophages, independent of gamma interferon (IFN-γ) activation. The ability of naive macrophages to clear type III parasites was dependent on enhanced activity of NADPH oxidase (Nox)-generated reactive oxygen species (ROS) and induction of guanylate binding protein 5 (Gbp5). Macrophages infected with type III parasites (CTG strain) showed a time-dependent increase in intracellular ROS generation that was higher than that induced by type I parasites (GT1 strain). The absence of Nox1 or Nox2, gp91 subunit isoforms of the Nox complex, reversed ROS-mediated clearance of CTG parasites. Consistent with this finding, both Nox1−/− and Nox2−/− mice showed higher susceptibility to CTG infection than wild-type mice. Additionally, Gbp5 expression was induced upon infection and the enhanced clearance of CTG strain parasites was reversed in Gbp5−/− macrophages. Expression of a type I ROP18 allele in CTG prevented clearance in naive macrophages, suggesting that it plays a role counteracting Gbp5. Although ROS and Gbp5 have been linked to activation of the NLRP3 inflammasome, clearance of CTG parasites did not rely on induction of pyroptosis. Collectively, these findings reveal that not all strains of T. gondii are adept at avoiding clearance in macrophages and define new roles for ROS and Gbps in controlling this important intracellular pathogen. IMPORTANCE Toxoplasma infections in humans and other mammals are largely controlled by IFN-γ produced by the activated adaptive immune system. However, we still do not completely understand the role of cell-intrinsic functions in controlling Toxoplasma or other apicomplexan infections. The present work identifies intrinsic activities in naive macrophages in counteracting T. gondii infection. Using an avirulent strain of T. gondii, we highlight the importance of Nox complexes in conferring protection against parasite infection both in vitro and in vivo. We also identify Gbp5 as a novel macrophage factor involved in limiting intracellular infection by avirulent strains of T. gondii. The rarity of human infections caused by type III strains suggests that these mechanisms may also be important in controlling human toxoplasmosis. These findings further extend our understanding of host responses and defense mechanisms that act to control parasitic infections at the cellular level.

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Affinity-Labeling of an NADPH-Binding Site on the Heavy Subunit of Flavocytochrome b558 in Particulate NADPH Oxidase from Activated Human Neutrophils

Biochemical and Biophysical Research Communications ◽

10.1006/bbrc.1993.2284 ◽

1993 ◽

Vol 196 (2) ◽

pp. 543-552 ◽

Cited By ~ 18

Author(s):

P. Ravel ◽

F. Lederer

Keyword(s):

Nadph Oxidase ◽

Binding Site ◽

Human Neutrophils ◽

Affinity Labeling ◽

Flavocytochrome B558

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Hypothyroidism-associated missense mutation impairs NADPH oxidase activity and intracellular trafficking of Duox2

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2014.05.006 ◽

2014 ◽

Vol 73 ◽

pp. 190-200 ◽

Cited By ~ 14

Author(s):

Ágnes Donkó ◽

Stanislas Morand ◽

Agnieszka Korzeniowska ◽

Howard E. Boudreau ◽

Melinda Zana ◽

...

Keyword(s):

Nadph Oxidase ◽

Missense Mutation ◽

Oxidase Activity ◽

Intracellular Trafficking ◽

Nadph Oxidase Activity

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Tumor necrosis factor-α in mechanic trauma plasma mediates cardiomyocyte apoptosis

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00578.2007 ◽

2007 ◽

Vol 293 (3) ◽

pp. H1847-H1852 ◽

Cited By ~ 30

Author(s):

Shuzhuang Li ◽

Xiangying Jiao ◽

Ling Tao ◽

Huirong Liu ◽

Yue Cao ◽

...

Keyword(s):

Nadph Oxidase ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Traumatic Injury ◽

Cardiac Injury ◽

Cardiomyocyte Apoptosis ◽

Therapeutic Interventions ◽

Tnf Α ◽

Ifn Γ ◽

Factor Α

Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation ( P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-α, IL-1β, and IFN-γ) or TNF-α alone, but not with IL-1β or IFN-γ alone, caused significant caspase-3 activation ( P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-α antibody, and TP isolated from TNF-α−/− mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-α (anti-TNF-α antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) ( P < 0.01). Taken together, our study demonstrated that there exists a TNF-α-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.

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S.129. The Role of Nuclear Factor Kappa B (NF-κB) and the IL-12/23-IFN-γ Axis in the Activation of the NADPH Oxidase System

Clinical Immunology ◽

10.1016/j.clim.2009.03.496 ◽

2009 ◽

Vol 131 ◽

pp. S167-S168

Author(s):

Walmir Aragão-Filho ◽

Juliana Moreira ◽

Marcos Luengo-Blanco ◽

Carolina Prando ◽

Jacinta Bustamante ◽

...

Keyword(s):

Nadph Oxidase ◽

Nuclear Factor Kappa B ◽

Nuclear Factor ◽

Oxidase System ◽

Nuclear Factor Kappa ◽

Ifn Γ

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High Ambient Temperature Aggravates Experimental Autoimmune Uveitis Symptoms

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.629306 ◽

2021 ◽

Vol 9 ◽

Author(s):

Su Pan ◽

Handan Tan ◽

Rui Chang ◽

Qingfeng Wang ◽

Ying Zhu ◽

...

Keyword(s):

Mass Spectrometry ◽

High Temperature ◽

Nadph Oxidase ◽

Ambient Temperature ◽

Th17 Cells ◽

Experimental Autoimmune Uveitis ◽

Temperature Group ◽

Ifn Γ ◽

Dependent Pathway ◽

Experimental Autoimmune

Whether ambient temperature influences immune responses leading to uveitis is unknown. We thus tested whether ambient temperature affects the symptoms of experimental autoimmune uveitis (EAU) in mice and investigated possible mechanisms. C57BL/6 mice were kept at a normal (22°C) or high temperature (30°C) housing conditions for 2 weeks and were then immunized with human interphotoreceptor retinoid-binding protein (IRBP651–670) peptide to induce EAU. Histological changes were monitored to evaluate the severity of uveitis. Frequency of Th1 cells and Th17 cells was measured by flow cytometry (FCM). The expression of IFN-γ and IL-17A mRNA was measured by real-time qPCR. The generation of neutrophil extracellular traps (NETs) was quantified by enzyme-linked immunosorbent assay (ELISA). Differential metabolites in the plasma of the mice kept in the aforementioned two ambient temperatures were measured via ultra-high-performance liquid chromatography triple quadrupole mass spectrometry quadrupole time of flight mass spectrometry (UHPLC-QQQ/MS). The differential metabolites identified were used to evaluate their effects on differentiation of Th1 and Th17 cells and generation of NETs in vitro. The results showed that EAU mice kept at high temperature experienced a more severe histopathological manifestation of uveitis than mice kept at a normal temperature. A significantly increased frequency of Th1 and Th17 cells in association with an upregulated expression of IFN-γ and IL-17A mRNA was observed in the splenic lymphocytes and retinas of EAU mice in high temperature. The expression of NETs as evidenced by myeloperoxidase (MPO) and neutrophil elastase (NE), was significantly elevated in serum and supernatants of neutrophils from EAU mice kept at high temperature compared to the normal temperature group. The metabolites in the plasma from EAU mice, fumaric acid and succinic acid, were markedly increased in the high temperature group and could induce the generation of NETs via the NADPH oxidase-dependent pathway, but did not influence the frequency of Th1 and Th17 cells. Our findings suggest that an increased ambient temperature is a risk factor for the development of uveitis. This is associated with the induction of Th1 and Th17 cells as well as the generation of NETs which could be mediated by the NADPH oxidase-dependent pathway.

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The Effect of IFN-γ and TNF-α on the NADPH Oxidase System of Human Colostrum Macrophages, Blood Monocytes, and THP-1 Cells

Journal of Interferon & Cytokine Research ◽

10.1089/jir.2005.25.540 ◽

2005 ◽

Vol 25 (9) ◽

pp. 540-546 ◽

Cited By ~ 12

Author(s):

Ana Carolina Almeida ◽

Jussara Rehder ◽

Silvana Dalgè Severino ◽

José Martins-Filho ◽

Peter E. Newburger ◽

...

Keyword(s):

Nadph Oxidase ◽

Blood Monocytes ◽

Oxidase System ◽

Tnf Α ◽

Human Colostrum ◽

Ifn Γ

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Interferon-γ activates transcription of NADPH oxidase 1 gene and upregulates production of superoxide anion by human large intestinal epithelial cells

AJP Cell Physiology ◽

10.1152/ajpcell.00135.2005 ◽

2006 ◽

Vol 290 (2) ◽

pp. C433-C443 ◽

Cited By ~ 75

Author(s):

Yuki Kuwano ◽

Tsukasa Kawahara ◽

Hironori Yamamoto ◽

Shigetada Teshima-Kondo ◽

Kumiko Tominaga ◽

...

Keyword(s):

Nadph Oxidase ◽

Tyrosine Phosphorylation ◽

Host Defense ◽

Intestinal Epithelium ◽

Superoxide Anion ◽

Promoter Activity ◽

Potential Contribution ◽

T84 Cells ◽

Nadph Oxidase 1 ◽

Ifn Γ

NADPH oxidase 1 (Nox1), a homolog of gp91phox, is dominantly expressed in large intestinal epithelium, and reactive oxygen species derived from Nox1 are suggested to serve a role in host defense. We report that interferon (IFN)-γ, a crucial transactivator of the gp91phoxgene, also stimulates expression of Nox1 mRNA and protein in large intestinal epithelium (T84 cells), leading to fourfold upregulation of superoxide anion (O2−) generation. Introduction of small interfering Nox1 RNA completely blocked this priming. We cloned the region from −4,831 to +195 bp of the human Nox1 gene. To reveal IFN-γ-responsive cis elements, we performed transient expression assays using a reporter gene driven by serially truncated Nox1 promoters in T84 cells. IFN-γ-responsive elements were located between −4.3 and −2.6 kb, and one γ-activated sequence (GAS) element present at −3,818 to −3,810 bp exhibited this IFN-γ-dependent promoter activity. IFN-γ caused tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1) and produced a protein-GAS complex that was recognized by anti-STAT1 antibody. The introduction of three-point mutation of GAS, which did not interact with STAT1, completely canceled the IFN-γ-dependent promoter activity of the region from −4,831 to +195 bp. A Janus protein tyrosine kinase 2 inhibitor (AG490) blocked the IFN-γ-stimulated tyrosine phosphorylation of STAT1, promoter activity of the −4,831 to +195 bp region, Nox1 mRNA expression, and O2−production, also suggesting a crucial role of STAT1 and GAS in the IFN-γ-stimulated transcription of the Nox1 gene. Our results support a potential contribution of Nox1 to mucosal host defense and inflammation in the colon.

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