Search

Impaired self-renewal of Kupffer cells (KCs) leads to inflammation in metabolic dysfunction-associated steatohepatitis (MASH). Here, we identify neutrophil cytosolic factor 1 (NCF1) as a critical regulator of iron homeostasis in KCs. NCF1 is upregulated in liver macrophages and dendritic cells in humans with metabolic dysfunction-associated steatotic liver disease and in MASH mice. Macrophage NCF1, but not dendritic cell NCF1, triggers KC iron overload, ferroptosis, and monocyte-derived macrophage infiltration, thus aggravating MASH progression. Mechanistically, elevated oxidized phospholipids induced by macrophage NCF1 promote Toll-like receptor (TLR4)-dependent hepatocyte hepcidin production, leading to increased KC iron deposition and subsequent KC ferroptosis. Importantly, the human low-functional polymorphic variant NCF1 90H alleviates KC ferroptosis and MASH in mice. In conclusion, macrophage NCF1 impairs iron homeostasis in KCs by oxidizing phospholipids, triggering hepatocyte hepcidin release and KC ferroptosis in MASH, highlighting NCF1 as a therapeutic target for improving KC fate and limiting MASH progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Aims: In this study, we investigate the role of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in autoimmune diseases. We focus on oxidative regulation at the interaction between antigen-presenting cells (APCs) and T cells, and consequent effect of ROS and RNS on type II collagen (CII)-induced arthritis (CIA) model in mice. Results: Mice deficient in ROS and peroxide, due to a mutation in Ncf1 gene, develop an exaggerated CIA and a stronger T cell response to CII. In contrast, nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was found to protect against CIA. The most pronounced protective effect was observed when L-NAME treatment started immediately after CII immunization. Ten days after immunization, the CII-reactive T cell-proliferative response was greater in Ncf1 -mutant mice that were treated with L-NAME. T cells from L-NAME-treated mice, primed with CII, showed lower interleukin-2 secretion in response to CII in vitro . Moreover, inhibition of RNS production resulted in dysregulation of NOS1 (neuronal) expression in CII-reactive T cells. Innovation and Conclusion: The results support that deficiency of a paracrine factor as ROS and peroxide released by APC leads to pronounced activation of T cells and enhanced arthritis. An intrinsic factor might be RNS produced by NOS1, which likely enhanced T cell activation in an autocrine manner.

Our previous study has demonstrated that 4-amino-2-trifluoromethyl-phenyl Retinate (ATPR) can induce human leukemia NB4 cells differentiation and G0/G1 phase arrest, but the underlying mechanism is still unclear. In this study, we used proteomics to screen differentially expressed protein profiles in NB4 cells before and after ATPR treatment in vitro. We analyzed the peptides digested from total cellular proteins by reverse phase LC-MS/MS and then performed label-free quantitative analysis. We found 27 significantly up-regulated proteins in the ATPR group compared to the control group. NCF1 was the most significantly changed protein. Immunoprecipitation and double immunofluorescent staining showed that EBP50 bind to NCF1. We further explored the potential molecular mechanism of EBP50/NCF1 complex in ATPR-induced differentiation and G0/G1 phase arrest. The results showed that ATPR remarkably reduced the expression of EBP50 in vivo and in vitro. Interestingly, the reduction of EBP50 contributed to ROS release by modulating the subcellular localization of NCF1. The reduction of EBP50 also contributed to G0/G1 phase arrest by inhibiting CyclinD1, CyclinA2 and CDK4, as well as promoting the differentiation of NB4 cells by increasing the expression of CD11b. Furthermore, we found that the overexpression of EBP50 restrained the effects of ATPR on differentiation and G0/G1 phase arrest in NB4 cells. These results suggest that ATPR-induced differentiation and G0/G1 phase arrest in acute promyelocytic leukemia (APL) by repressing EBP50/NCF1 complex to promote the production of ROS, and the results from in vivo experiments were consistent with those from in vitro studies. Therefore, our finding results suggest that EBP50 may be a new target for ATPR in the treatment of APL., (Copyright © 2019. Published by Elsevier Inc.)

Background: Increased reactive oxygen species and estrogen deficiency contribute to the pathophysiology of postmenopausal osteoporosis. Reactive oxygen species contribute to bone degradation and is necessary for RANKL-induced osteoclast differentiation. In postmenopausal bone loss, reactive oxygen species can also activate immune cells to further enhance bone resorption. Here, we investigated the role of reactive oxygen species in ovariectomy-induced osteoporosis in mice deficient in Ncf1, a subunit for the NADPH oxidase 2 and a well-known regulator of the immune system., Methods: B10.Q wild-type (WT) mice and mice with a spontaneous point mutation in the Ncf1-gene (Ncf1*/*) were ovariectomized (ovx) or sham-operated. After 4 weeks, osteoclasts were generated ex vivo, and bone mineral density was measured using peripheral quantitative computed tomography. Lymphocyte populations, macrophages, pre-osteoclasts and intracellular reactive oxygen species were analyzed by flow cytometry., Results: After ovx, Ncf1*/*-mice formed fewer osteoclasts ex vivo compared to WT mice. However, trabecular bone mineral density decreased similarly in both genotypes after ovx. Ncf1*/*-mice had a larger population of pre-osteoclasts, whereas lymphocytes were activated to the same extent in both genotypes., Conclusion: Ncf1*/*-mice develop fewer osteoclasts after ovx than WT mice. However, irrespective of genotype, bone mineral density decreases after ovx, indicating that a compensatory mechanism retains bone degradation after ovx.

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs). [ABSTRACT FROM AUTHOR]

Unexplained recurrent spontaneous abortion (URSA) is a major concern in reproductive medicine. Neutrophil cytosolic factor 1 (NCF1) polymorphisms leading to low production of reactive oxygen species (ROS) are strongly associated with autoimmune diseases. We investigated the association of the missense single nucleotide polymorphism (SNP) rs201802880 (NCF1-339) in NCF1 with URSA and explored its function. We performed NCF1-339 SNP genotyping of samples from 152 Chinese patients with URSA and 72 healthy controls using nested PCR and TaqMan assays. ROS production and RELA (NF-κB subunit) expression in the blood of participants with different NCF1-339 genotypes were determined. The frequencies of the wild-type (GG) and mutant (GA) genotypes remarkably differed between the URSA and control groups. The mutant genotype was associated with an increased risk of recurrent abortion. Furthermore, ROS levels in the URSA group with the GG genotype were significantly higher than those in the group with the GA genotype (p < 0.05). RELA expression in URSA patients with the GA genotype was considerably higher than that in control individuals with the GG genotype. These findings indicate that mutations in NCF1 may increase the risk of URSA via the NADP/ROS/NF-κB signaling pathway, which has implications for the diagnosis and treatment of URSA.

The positional cloning of single nucleotide polymorphisms (SNPs) of the neutrophil cytosolic factor 1 (Ncf1) gene, advocating that a low oxidative burst drives autoimmune disease, demands an understanding of the underlying molecular causes. A cellular target could be T cells, which have been shown to be regulated by reactive oxygen species (ROS). However, the pathways by which ROS mediate T cell signaling remain unclear. The adaptor molecule linker for activation of T cells (LAT) is essential for coupling T cell receptor-mediated antigen recognition to downstream responses, and it contains several cysteine residues that have previously been suggested to be involved in redox regulation. To address the possibility that ROS regulate T cell-dependent inflammation through LAT, we established a mouse strain with cysteine-to-serine mutations at positions 120 and 172 (LATSS). We found that redox regulation of LAT through C120 and C172 mediate its localization and phosphorylation. LATSS mice had reduced numbers of double-positive thymocytes and naïve peripheral T cells. Importantly, redox insensitivity of LAT enhanced T cell-dependent autoimmune inflammation in collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). This effect was reversed on an NCF1-mutated (NCF1m1j), ROS-deficient, background. Overall, our data show that LAT is redox-regulated, acts to repress T cell activation, and is targeted by ROS induced by NCF1 in antigen-presenting cells (APCs).

Traumatic brain injury (TBI) is a predominant cause of long-term disability in adults, yet the molecular mechanisms underpinning the neuropathological processes associated with it remain inadequately understood. Neutrophil cytosolic factor 1 (NCF1, also known as p47phox) is one of the cytosolic components of NADPH oxidase NOX2. In this study, we observed a reduction in the volume of TBI-induced brain lesions in NCF1-knockout mice compared to controls. Correspondingly, the neuronal loss induced by TBI was mitigated in the NCF1-knockout mice. Behavioral analysis also demonstrated that the motor coordination deficit following TBI was mitigated by the depletion of NCF1. Mechanistically, our findings revealed that NCF1 deficiency attenuated TBI-induced inflammatory responses by inhibiting the release of proinflammatory factors and reducing neutrophil infiltration into the brain parenchyma. Additionally, our results indicated that NCF1 deficiency significantly decreased the levels of reactive oxygen species in neutrophils. Taken together, our findings indicate that NCF1 plays a crucial role in the regulation of brain injury and secondary inflammation post-TBI. • NCF1 deficiency attenuated TBI-induced inflammatory responses. • NCF1 deficiency reduced neutrophil infiltration into the brain parenchyma. • NCF1 deficiency inhibited the release of proinflammatory factors. • NCF1 deficiency significantly decreased the levels of reactive oxygen species in neutrophils. [ABSTRACT FROM AUTHOR]

Aims: Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in non-obese diabetic (NOD) mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of Ncf1 (the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC).Results:Ncf1-mutant DCs exhibit reduced ability to activate autoreactive CD8+ T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen, Ncf1-mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules.Innovation: This study demonstrates that Ncf1 (p47phox) is required for activation and effector function of CD8+ T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells.Conclusion: ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8+ T cells in individuals at-risk for developing T1D.

To identify differentially expressed genes in sepsis and potential key role of reactive oxygen species (ROS) genes associated with sepsis. Gene expression dataset was available from GSE46599. Firstly, we screened the differentially expressed genes between sepsis and healthy samples. Then, the Database for Annotation, Visualization and Integrated Discovery (DAVID) online tools were utilized to perform gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses at the functional level. Differentially expressed genes mediating ROS levels were validated in the next investigation and analysis. We identified 1094 genes expressed differentially between normal and sepsis samples, including 655 upregulated genes and 439 downregulated genes. At the functional level, GO and KEGG pathway enrichment analysis showed that those differentially expressed genes were majorly associated with the immune response and metabolic process in sepsis. Further analysis revealed that neutrophil cytosolic factor 1(NCF1), a critical gene in the ROS system, upregulated in THP-1 cell and monocytes under lipopolysaccharides stimulation. Moreover, we identified the upregulation of NCF1 in a sepsis model. We screened the differentially expressed genes from the global level and identified NCF1 might be a critical target gene in sepsis. [ABSTRACT FROM AUTHOR]

Aims: Reactive oxygen species (ROS) are critical in driving the onset of type 1 diabetes (T1D). Ablation of ROS derived from phagocytic NADPH oxidase 2 is protective against autoimmune diabetes in non-obese diabetic (NOD) mice. However, the mechanisms of NADPH oxidase 2-derived ROS in T1D pathogenesis need to be elucidated. Here, we have examined the role of Ncf1 (the regulatory subunit of NADPH oxidase 2) in dendritic cells (DC). Results: Ncf1 -mutant DCs exhibit reduced ability to activate autoreactive CD8+ T cells despite no difference in co-stimulatory molecule expression or pro-inflammatory cytokine production. When provided with exogenous whole-protein antigen, Ncf1 -mutant NOD DCs showed strong phagosome acidification and rapid antigen degradation, which lead to an absence of protein translocation into the cytoplasm and deficient antigenic peptide loading on MHC Class I molecules. Innovation: This study demonstrates that Ncf1 (p47phox) is required for activation and effector function of CD8+ T cells by acting both intrinsically within the T cell as well as within professional antigen presenting cells. Conclusion: ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation by DCs. ROS scavengers could potentially represent an important component of therapies aiming to disrupt autoantigen presentation and activation of CD8+ T cells in individuals at-risk for developing T1D. [ABSTRACT FROM AUTHOR]

The current review on the function of neutrophil cytosolic factor 1 (NCF1) and induced reactive oxygen species ( ROS) is based on a genetic search for the major genes controlling autoimmune inflammatory disorders. Surprisingly, the disease-promoting allele determined a lower ROS response and was therefore in complete contrast to the prevailing dogma. Once cloned, it opened the possibility to dissect this complex field from a new angle and with the possibilities to study the role of ROS in vivo. We found that NCF1 and NADPH oxidase 2 ( NOX2) complex-derived ROS is an important regulator of several chronic inflammatory disorders by using models for rheumatoid arthritis, multiple sclerosis, psoriasis and psoriasis arthritis, gout, and lupus. ROS could therefore affect many different types of diseases and the common denominator seems to be that ROS regulate macrophages, which prevents inflammation from going chronic. The role of ROS is currently changing from being seen as toxic agents that will promote inflammation toward a more complex view with ROS as crucial regulators of immune and inflammatory pathways. [ABSTRACT FROM AUTHOR]

Multiple sclerosis (MS) is pathologically characterized by inflammatory demyelination and neuronal injury. Although phagocytosis of myelin debris by microglia and macrophages in acute MS lesions is well documented, its pathophysiological significance is unclear. Using real-time quantitative PCR, flow cytometry, ELISA, and reactive oxygen species (ROS) measurement assays, we demonstrated that phagocytosis of myelin modulates activation of microglial cells prestimulated by interferon-γ(IFN-γ) or a combination of IFN-γ and lipopolysaccharide with a biphasic temporal pattern, i.e., enhanced production of proinflammatory mediators during the first phase (≤6 h), followed by suppression during the second (6-24 h) phase. In this second phase, myelin phagocytosis leads to an enhanced release of prostaglandin E2 and ROS in microglia, whereas the production of anti-inflammatory cytokines (particularly interleukin-10) remains unchanged. Suppression of inflammatory microglial activation by myelin phagocytosis was reversed by treatment with superoxide dismutase and catalase, by inhibition of the NADPH-oxidase complex, or by specific knockdown of the NADPH-oxidase-required adaptor p47-phagocyte oxidase (PHOX). Furthermore, we observed that myelin phagocytosis destabilized tumor necrosis factor-α and interferon-induced protein-10 mRNA through an adenine- uridine-rich elements-involved mechanism, which was reversed by blocking the function of NADPH-oxidase complex. We conclude that phagocytosis of myelin suppresses microglial inflammatory activities via enhancement of p47-PHOX-mediated ROS generation. These results suggest that intervention in ROS generation could represent a novel therapeutic strategy to reduce neuroinflammation in MS. [ABSTRACT FROM AUTHOR]

Background: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency of the phagocytic cells, which results in absent or diminished levels of microbicidal reactive oxygen species. The disease occurs due to germline mutations in the genes encoding the five subunits of NADPH oxidase complex. The present study is a pilot study to understand the clinical and genetic aspects of CGD in Sri Lanka. Methods: Clinical records of thirteen CGD patients were analysed and compared with similar studies performed in different countries and regions to identify patterns in demographics, clinical manifestations and infectious agents. Genomic DNA and cDNA were analysed in eight patients to identify mutations in CYBB and NCF1 genes, thereby to ascertain the potential X-linked and autosomal recessive (AR) CGD patients. Results: The onset of symptoms in the patient cohort was very early (mean 4.6 months) compared to 20 months in India and 23.9 months in Latin America. Similarly, the age at diagnosis was lower (mean 1.6 years after birth) compared to other studies; 4.5 years in India and 6.1 years in Europe. Pulmonary manifestations were the most common (85%), followed by skin/subcutaneous infections (77%) and lymphadenopathy (62%). The death rate of local patients (38%) was higher than other countries (India 35%, Europe 20%). Majority (77%) were treated for tuberculosis at some point in life. Genetic analysis confirmed six out of eight patients as X-linked CGD cases with mutations in CYBB gene. A novel splice site mutation was identified in P-07 at position c.141+6 which resulted in the deletion of entire exon 2. Two siblings (P-05 and P-06) from consanguineous parents, were identified with AR-CGD based on the homozygous GT deletion mutation in NCF1 gene. Conclusions: The clinical presentation, manifestations and genetic subtypes in the local cohort, appear to be comparable with global trends. Mycobacterial infections should be investigated and treated with more prominence. Effective treatment options are required to control the high mortality rate. [ABSTRACT FROM AUTHOR]