Stimulation of cells with various peptide growth factors induces the production of phosphatidylinositol 3,4,5-trisphosphate ( PIP3) through activation of phosphatidylinositol 3-kinase. The action of ...this enzyme is reversed by that of the tumor suppressor PTEN. With the use of cells overexpressing NADPH oxidase 1 or peroxiredoxin II, we have now shown that H2O2produced in response to stimulation of cells with epidermal growth factor or platelet-derived growth factor potentiates PIP3generation and activation of the protein kinase Akt induced by these growth factors. We also show that a small fraction of PTEN molecules is transiently inactivated as a result of oxidation of the essential cysteine residue of this phosphatase in various cell types stimulated with epidermal growth factor, platelet-derived growth factor, or insulin. These results suggest that the activation of phosphatidylinositol 3-kinase by growth factors might not be sufficient to induce the accumulation of PIP3because of the opposing activity of PTEN and that the concomitant local inactivation of PTEN by H2O2might be needed to increase the concentration of PIP3sufficiently to trigger downstream signaling events. Furthermore, together with previous observations, our data indicate that peroxiredoxin likely participates in PIP3signaling by modulating the local concentration of H2O2.
Despite the advancements in vaccination research and practices, influenza viruses remain a global health concern. Inducing a robust immune response by vaccination is especially challenging in the ...elderly, the immunocompromised, and persons with chronic illnesses. Polysaccharides derived from food may act as a safe and readily accessible means to boost the immune system during vaccination. In this study, we investigated whether crude polysaccharides derived from carrot pomace (CPP) could stimulate innate immune cell function and promote influenza vaccine immunogenicity. In bone marrow-derived dendritic cells (BMDCs), CPP increased the fraction of CD11c+MHCII+ cells and the expression of co-stimulatory molecules CD40 and CD80, indicative of enhanced maturation and activation. Functionally, CPP-treated BMDCs promoted inflammatory cytokine production in splenic lymphocytes. In a mouse model of immunosuppression induced by cyclophosphamide, animals given CPP before and after an influenza vaccine challenge showed increased frequencies of dendritic cells and natural killer cells in the spleen, in addition to the recovery of vaccine-specific antibody titers. Moreover, innate myeloid cells in CPP-fed mice showed evidence of phenotypic modification via markedly enhanced interleukin(IL)-12 and interferon(IFN)-γ production in response to lipopolysaccharide(LPS) stimulation ex vivo. Our findings suggest that the administration of carrot pomace polysaccharides can significantly enhance the efficacy of influenza vaccination.
The tumor suppressor PTEN regulates cell migration, growth, and survival by removing the 3′-phosphate of phosphoinositides. Exposure of purified PTEN or of cells to H2O2 resulted in inactivation of ...PTEN in a time- and H2O2concentration-dependent manner. Analysis of various cysteine mutants, including mass spectrometry of tryptic peptides, indicated that the essential Cys124 residue in the active site of PTEN specifically forms a disulfide with Cys71during oxidation by H2O2. The reduction of H2O2-oxidized PTEN in cells appears to be mediated predominantly by thioredoxin. Thus, thioredoxin was more efficient than glutaredoxin, glutathione, or a 14-kDa thioredoxin-like protein with regard to the reduction of oxidized PTEN in vitro. Thioredoxin co-immunoprecipitated with PTEN from cell lysates; and incubation of cells with 2,4-dinitro-1-chlorobenzene (an inhibitor of thioredoxin reductase) delayed the reduction of oxidized PTEN, whereas incubation with buthioninesulfoximine (an inhibitor of glutathione biosynthesis) did not. These results suggest that the reversible inactivation of PTEN by H2O2might be important for the accumulation of 3′-phosphorylated phosphoinositides and that the uncontrolled generation of H2O2 associated with certain pathological conditions might contribute to cell proliferation by inhibiting PTEN function.
Nox1 is an abundant source of reactive oxygen species (ROS) in colon epithelium recently shown to function in wound healing and epithelial homeostasis. We identified Peroxiredoxin 6 (Prdx6) as a ...novel binding partner of Nox activator 1 (Noxa1) in yeast two-hybrid screening experiments using the Noxa1 SH3 domain as bait. Prdx6 is a unique member of the Prdx antioxidant enzyme family exhibiting both glutathione peroxidase and phospholipase A2 activities. We confirmed this interaction in cells overexpressing both proteins, showing Prdx6 binds to and stabilizes wild type Noxa1, but not the SH3 domain mutant form, Noxa1 W436R. We demonstrated in several cell models that Prdx6 knockdown suppresses Nox1 activity, whereas enhanced Prdx6 expression supports higher Nox1-derived superoxide production. Both peroxidase- and lipase-deficient mutant forms of Prdx6 (Prdx6 C47S and S32A, respectively) failed to bind to or stabilize Nox1 components or support Nox1-mediated superoxide generation. Furthermore, the transition-state substrate analogue inhibitor of Prdx6 phospholipase A2 activity (MJ-33) was shown to suppress Nox1 activity, suggesting Nox1 activity is regulated by the phospholipase activity of Prdx6. Finally, wild type Prdx6, but not lipase or peroxidase mutant forms, supports Nox1-mediated cell migration in the HCT-116 colon epithelial cell model of wound closure. These findings highlight a novel pathway in which this antioxidant enzyme positively regulates an oxidant-generating system to support cell migration and wound healing.
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•Peroxiredoxin6 (Prdx6) binds to the SH3 domain of Nox activator 1.•Prdx6 assembles with the NADPH oxidase 1 (Nox1) complex.•Prdx6 expression enhances Nox1-derived superoxide production.•The Nox1-supportive effects of Prdx6 involve its phospholipase A2 activity.•Wild-type Prdx6 supports Nox1-mediated migration of colon epithelial cells.
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.
Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Nox) are an important family of catalytic enzymes that generate reactive oxygen species (ROS), which mediate the regulation of diverse ...cellular functions. Although phagocyte Nox2/gp91phox is closely associated with the activation of host innate immune responses, the roles of Nox family protein during Toxoplasma gondii (T. gondii) infection have not been fully investigated. Here, we found that T. gondii-mediated ROS production was required for the upregulation of macrophage migration inhibitory factor (MIF) mRNA and protein levels via activation of mitogen-activated protein kinase and nuclear factor-κB signaling in macrophages. Interestingly, MIF knockdown led to a significant increase in the survival of intracellular T. gondii in bone marrow-derived macrophages (BMDMs). Moreover, Nox4 deficiency, but not Nox2/gp91phox and the cytosolic subunit p47phox, resulted in enhanced survival of the intracellular T. gondii RH strain and impaired expression of T. gondii-mediated MIF in BMDMs. Additionally, Nox4-deficient mice showed increased susceptibility to virulent RH strain infection and increased cyst burden in brain tissues and low levels of MIF expression following infection with the avirulent ME49 strain. Collectively, our findings indicate that Nox4-mediated ROS generation plays a central role in MIF production and resistance to T. gondii infection.
The physiological effects of the many germline mutations of TP53, encoding the tumor suppressor protein p53, are poorly understood. Here we report generating a p53 R178C knockin mouse modeling the ...human TP53 R181C mutation, which is notable for its prevalence and prior molecular characterization. Consistent with its weak cancer penetrance in humans, homozygous p53178C/C mice show a modest increase in tumorigenesis but, surprisingly, are lean with decreased body fat content. They display evidence of increased lipolysis and upregulation of fatty acid metabolism in their inguinal white adipose tissue (iWAT). Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses show that the mutant p53 bound and transactivated Beta-3-Adrenergic Receptor (ADRB3), a gene that is known to promote lipolysis and is associated with obesity. This study reveals that a germline mutation of p53 can affect fat metabolism, which has been implicated in cancer development.
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•Mouse homolog of a TP53 germline mutation reveals a metabolic phenotype•Mutant p53 R178C differentially retains wild-type p53 activity•ChIP-seq analysis identifies adrenergic receptor ADRB3 as a p53 target gene•p53 R178C promotes lipolysis in adipocytes by increased ADRB3 signaling
Knockin of the mouse homolog of a human TP53 germline mutation known to cause Li-Fraumeni syndrome, a cancer predisposition disorder, results in a mouse model characterized by lower body fat content. Kang et al. show that enhancing transactivation of the lipolytic gene ADRB3 by mutant p53 contributes to this phenotype.
Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of ...each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells.
Rheumatoid arthritis (RA) is a common autoimmune disease characterized by immune cell infiltration of the synovium, leading to the loss of cartilage, bone, and joint function. Although regulatory T ...(Treg) cells are thought to modulate the initiation and progression of RA, a consensus has yet to be reached regarding the function and composition of Treg cells in RA patients. To address these discrepancies, we analyzed not only the total Treg frequency but also that of Treg subpopulations in the peripheral blood of RA patients and healthy controls by flow cytometry. We found that the total Treg population was not significantly different between RA and control subjects. However, the effector Treg cell subgroup, defined as CD45RA−CD25hi, showed markedly decreased frequency in RA patients. In addition, the total Treg population from RA patients showed a significant decline in the expression of CD25. Both the naïve and effector Treg subgroups also showed marked reduction of CD25 expression in RA patients compared to controls. These data suggest that the decreased frequency of effector Treg cells and overall reduction of CD25 expression in Treg cells in the peripheral blood may be evidence of altered Treg homeostasis associated with RA pathogenesis.
Autoimmune limbic encephalitis (LE) is a rare, but devastating complication of allogeneic hematopoietic stem cell transplantation (HSCT). There is currently limited evidence describing the risk ...factors, laboratory features, and underlying mechanisms of this neurologic adverse event. We retrospectively reviewed available clinical, imaging, and laboratory data from adult patients with hematological malignancies who underwent haploidentical HSCT with post-transplant cyclophosphamide (PTCy) at Chungnam National University Hospital from June 2016 to May 2020. Patients who developed LE were compared to those who did not based on clinical assessment, serum inflammatory biomarkers, and reconstitution of various T cell populations. Of 35 patients, 4 developed LE. There were no differences in patient demographics, donor demographics, or treatment conditions between patients that did and did not develop LE. Overall, patients with LE had worse clinical outcomes and overall survival than those without. In addition, they tended to have higher markers of systemic inflammation in the early post-transplant period, including fever, C-reactive protein (CRP), and cytokines. Remarkably, baseline interleukin-6 levels before HSCT were found to be higher in patients who developed LE than those who did not. In addition, analysis of T cell subsets showed impaired expansion of CD25+FOXP3+ regulatory T (Treg) cells in LE compared to non-LE patients despite appropriate reconstitution of the total CD4+ T cell population. Patients that developed LE within the first 30 days of HSCT were likely to have high serum IL-6 among other inflammatory cytokines coupled with suppression of regulatory T cell differentiation. Further work is needed on the mechanisms underlying impaired Treg expansion following HSCT and potential therapies.