Construction of three-gene-based prognostic signature and analysis of immune cells infiltration in children and young adults with B-acute lymphoblastic leukemia.

BACKGROUND: Although B-acute lymphoblastic leukemia (B-ALL) patients' survival has been improved dramatically, some cases still relapse. This study aimed to explore the prognosis-related novel differentially expressed genes (DEGs) for predicting the overall survival (OS) of children and young adults (CAYAs) with B-ALL and analyze the immune-related factors contributing to poor prognosis. METHODS: GSE48558 and GSE79533 from Gene Expression Omnibus (GEO) and clinical sample information and mRNA-seq from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database were retrieved. Prognosis-related key genes were enrolled to build a Cox proportional model using multivariate Cox regression. Five-year OS of patients, clinical characteristic relevance and clinical independence were assessed based on the model. The mRNA levels of prognosis-related genes were validated in our samples and the difference of immune cells composition between high-risk and low-risk patients were compared. RESULTS: One hundred and twelve DEGs between normal B cells and B-ALL cells were identified based on GSE datasets. They were mainly participated in protein binding and HIF-1 signaling pathway. One hundred and eighty-nine clinical samples were enrolled in the study, both Kaplan-Meier (KM) analysis and univariate Cox regression analysis showed that CYBB, BCL2A1, IFI30, and EFNB1 were associated with prognosis, CYBB, BCL2A1, and EFNB1 were used to construct prognostic risk model. Moreover, compared to clinical indicators, the three-gene signature was an independent prognostic factor for CAYAs with B-ALL. Finally, the mRNA levels of CYBB, BCL2A1, and EFNB1 were significantly lower in B-ALL group as compared to controls. The high-risk group had a significantly higher percentage of infiltrated immune cells. CONCLUSION: We constructed a novel three-gene signature with independent prognostic factor for predicting 5-year OS of CAYAs with B-ALL. Additionally, we discovered the difference of immune cells composition between high-risk and low-risk groups. This study may help to customize individual treatment and improve prognosis of CAYAs with B-ALL.

C1q/tumour necrosis factor-related protein-9 aggravates lipopolysaccharide-induced inflammation via promoting NLRP3 inflammasome activation.

The NLRP3 inflammasome plays a vital role in inflammation by increasing the maturation of interleukin-1ß (IL-1ß) and promoting pyroptosis. Given that C1q/tumour necrosis factor-related protein-9 (CTRP9) has been shown to be involved in diverse inflammatory diseases, we sought to assess the underlying impact of CTRP9 on NLRP3 inflammasome activation. In vitro, macrophages isolated from murine peritonea were stimulated with exogenous CTRP9, followed by lipopolysaccharide (LPS) and adenosine 5'-triphosphate (ATP). We demonstrated that CTRP9 markedly augmented the activation of the NLRP3 inflammasome, as shown by increased mature IL-1ß secretion, triggering ASC speck formation and promoting pyroptosis. Mechanistically, CTRP9 increased the levels of NADPH oxidase 2 (NOX2)-derived reactive oxygen species (ROS). Suppressing ROS with N-acetylcysteine (NAC) or interfering with NOX2 by small interfering RNA weakened the promoting effect of CTRP9 on the NLRP3 inflammasome. Furthermore, NLRP3 inflammasome activation, pyroptosis and secretion of mature IL-1ß were significantly decreased in macrophages from CTRP9-KO mice compared to those from WT mice with the same treatment. In vivo, we established a sepsis model by intraperitoneal injection of LPS into WT and CTRP9-KO mice. CTRP9 knockout improved the survival rates of the septic mice and attenuated NLRP3 inflammasome-mediated inflammation. In conclusion, our study indicates that CTRP9 aggravates LPS-induced inflammation by promoting NLRP3 inflammasome activation via the NOX2/ROS pathway. CTRP9 could be a promising target for NLRP3 inflammasome-driven inflammatory diseases.

Fine definition of the epitopes on the human gp91phox/NOX2 for the monoclonal antibodies CL-5 and 48.

Superoxide-producing NADPH oxidase, gp91phox/NOX2, in phagocytes plays a critical role in the host defenses against pathogens. Moreover, gp91phox/NOX2 contributes to the oxidative stress in endothelial cells. Therefore, investigating the level of gp91phox/NOX2 with immunoblotting is important for estimating the amount of superoxide produced. Here, we showed that the epitopes in human gp91phox/NOX2 recognized by monoclonal antibodies (mAbs) CL-5 and 48 were in amino acids 132-147 and 136-144, respectively. Although the epitopes were close to the N-glycosylation sites, N-glycan maturation did not affect mAbs recognition. When Pro-136 was substituted with Arg, the corresponding mouse residue, human gp91phox/NOX2 was not recognized by mAbs CL-5 and 48; however, the substitution did not affect gp91phox/NOX2-based oxidase activity. This finding explains why these mAbs specifically recognize the human but not mouse gp91phox/NOX2. Hence, these mAbs are useful for investigating the level of gp91phox/NOX2 without amino acid substitutions in the epitopes.

Beyond the Extra Respiration of Phagocytosis: NADPH Oxidase 2 in Adaptive Immunity and Inflammation.

Reactive oxygen species (ROS) derived from the phagocyte NADPH oxidase (NOX2) are essential for host defence and immunoregulation. Their levels must be tightly controlled. ROS are required to prevent infection and are used in signalling to regulate several processes that are essential for normal immunity. A lack of ROS then leads to immunodeficiency and autoinflammation. However, excess ROS are also deleterious, damaging tissues by causing oxidative stress. In this review, we focus on two particular aspects of ROS biology: (i) the emerging understanding that NOX2-derived ROS play a pivotal role in the development and maintenance of adaptive immunity and (ii) the effects of excess ROS in systemic disease and how limiting ROS might represent a therapeutic avenue in limiting excess inflammation.

NOX2-Deficient Neutrophils Facilitate Joint Inflammation Through Higher Pro-Inflammatory and Weakened Immune Checkpoint Activities.

Immune-mediated arthritis is an important chronic inflammatory disease of joints causing debilitating morbidity in affected patients. The mechanisms underlying immune-mediated arthritis have been intensively investigated, however the cellular and molecular factors contributing to the joint inflammation in different redox conditions have not been clearly elucidated. Previous research showed that phagocyte-produced reactive oxygen species (ROS) plays an anti-inflammatory role in K/BxN serum-transfer arthritis and NOX2-deficient mice tend to have more severe arthritis. Although many leukocytes play critical roles in the development of immune-mediated arthritis, the role of neutrophils, which are the main producers of ROS in inflammation, is still controversial. We hence assessed the immunomodulatory function of neutrophils from arthritic joints of NOX2-deficient and wild type mice in this study. We found more neutrophils accumulation in NOX2-deficient inflamed joints. RNA-sequencing and quantitative PCR revealed significantly increased expression of acute inflammation genes including IL1b, Cxcl2, Cxcl3, Cxcl10 and Mmp3 in activated neutrophils from the inflamed joints of NOX2-deficient mice. Moreover, gene set enrichment analysis (GSEA) showed enriched gene signatures in type I and II IFN responses, IL-6-JAK-STAT3 signaling pathway and TNF-α signaling pathway via NF-κB in NOX2-deficient neutrophils. In addition, we found that NOX2-deficient neutrophils expressed lower levels of PD-L1 and were less suppressive than WT neutrophils. Moreover, treatment of PD-L1-Fc decreased cytokine expression and ameliorated the severity of inflammatory arthritis. Our results suggest that NOX2-derived ROS is critical for regulating the function and gene expression in arthritic neutrophils. Both the strong pro-inflammatory and weakened anti-inflammatory functions of neutrophils due to abnormal redox regulation may be targets of treatment for immune-mediated arthritis.

Rac-dependent feedforward autoactivation of NOX2 leads to oxidative burst.

NADPH oxidase 2 (NOX2) produces the superoxide anion radical (O2-), which has functions in both cell signaling and immune defense. NOX2 is a multimeric-protein complex consisting of several protein subunits including the GTPase Rac. NOX2 uniquely facilitates an oxidative burst, which is described by initially slow O2- production, which increases over time. The NOX2 oxidative burst is considered critical to immune defense because it enables expedited O2- production in response to infections. However, the mechanism of the initiation and progression of this oxidative burst and its implications for regulation of NOX2 have not been clarified. In this study, we show that the NOX2 oxidative burst is a result of autoactivation of NOX2 coupled with the redox function of Rac. NOX2 autoactivation begins when active Rac triggers NOX2 activation and the subsequent production of O2-, which in turn activates redox-sensitive Rac. This activated Rac further activates NOX2, amplifying the feedforward cycle and resulting in a NOX2-mediated oxidative burst. Using mutagenesis-based kinetic and cell analyses, we show that enzymatic activation of Rac is exclusively responsible for production of the active Rac trigger that initiates NOX2 autoactivation, whereas redox-mediated Rac activation is the main driving force of NOX2 autoactivation and contributes to generation of ∼98% of the active NOX2 in cells. The results of this study provide insight into the regulation of NOX2 function, which could be used to develop therapeutics to control immune responses associated with dysregulated NOX2 oxidative bursts.

TREM2 Dictates Antibacterial Defense and Viability of Bone Marrow-derived Macrophages during Bacterial Infection.

Macrophages undergo profound metabolic reprogramming to join key immunoregulatory functions, which can be initiated by pattern recognition receptors. TREM2 (triggering receptor expressed on myeloid cells 2), a macrophage phagocytic receptor, plays pivotal roles in sepsis by enhancing bacterial clearance, which is associated with regulation of reactive oxygen species (ROS) production. However, how intracellular ROS participate in TREM2-mediated bactericidal activity remains unclear. This study was designed to investigate the organelle source and biological activity of ROS in the context of TREM2-mediated immune defense during Escherichiacoli infection. Bone marrow-derived macrophages (BMDMs) were transfected with TREM2-overexpressing adenoviruses or control viruses and challenged with E. coli. The BMDMs were administered to mouse models with local E. coli infection. In addition, monocytic TREM2 expression, NOX2 concentrations, and pyroptosis were detected in patients with bacterial sepsis. General ROS production was found to be comparable between TREM2-overexpressing and control BMDMs upon E. coli challenge. The deficiency of Nox2 led to impaired phagosome degradation and lack of bactericidal ability and abolished TREM2-mediated protective activity against pulmonary E. coli infection. Overexpression of TREM2 suppressed mitochondrial ROS generation, inhibited NLRP3/caspase-1 inflammasome activation, and finally protected BMDMs from gasdermin D-mediated pyroptosis during pulmonary E. coli infection. The protective role of TREM2 was further confirmed in mice with abdominal E. coli infection. Moreover, monocytic TREM2 expression was positively correlated with NOX2 concentrations and negatively correlated with pyroptosis and disease severity in patients with bacterial sepsis. Collectively, TREM2 controls macrophage immune functions by fine-tuning ROS generation and enhances the host defense against bacterial infection. Our data suggest that TREM2 is a promising candidate target for sepsis therapy.

Microglial Nox2 Plays a Key Role in the Pathogenesis of Experimental Autoimmune Encephalomyelitis.

While oxidative stress has been linked to multiple sclerosis (MS), the role of superoxide-producing phagocyte NADPH oxidase (Nox2) in central nervous system (CNS) pathogenesis remains unclear. This study investigates the impact of Nox2 gene ablation on pro- and anti-inflammatory cytokine and chemokine production in a mouse experimental autoimmune encephalomyelitis (EAE) model. Nox2 deficiency attenuates EAE-induced neural damage and reduces disease severity, pathogenic immune cells infiltration, demyelination, and oxidative stress in the CNS. The number of autoreactive T cells, myeloid cells, and activated microglia, as well as the production of cytokines and chemokines, including GM-CSF, IFNγ, TNFα, IL-6, IL-10, IL-17A, CCL2, CCL5, and CXCL10, were much lower in the Nox2-/- CNS tissues but remained unaltered in the peripheral lymphoid organs. RNA-seq profiling of microglial transcriptome identified a panel of Nox2 dependent proinflammatory genes: Pf4, Tnfrsf9, Tnfsf12, Tnfsf13, Ccl7, Cxcl3, and Cxcl9. Furthermore, gene ontology and pathway enrichment analyses revealed that microglial Nox2 plays a regulatory role in multiple pathways known to be important for MS/EAE pathogenesis, including STAT3, glutathione, leukotriene biosynthesis, IL-8, HMGB1, NRF2, systemic lupus erythematosus in B cells, and T cell exhaustion signaling. Taken together, our results provide new insights into the critical functions performed by microglial Nox2 during the EAE pathogenesis, suggesting that Nox2 inhibition may represent an important therapeutic target for MS.

NOTCH-induced rerouting of endosomal trafficking disables regulatory T cells in vasculitis.

The aorta and the large conductive arteries are immunoprivileged tissues and are protected against inflammatory attack. A breakdown of immunoprivilege leads to autoimmune vasculitis, such as giant cell arteritis, in which CD8+ Treg cells fail to contain CD4+ T cells and macrophages, resulting in the formation of tissue-destructive granulomatous lesions. Here, we report that the molecular defect of malfunctioning CD8+ Treg cells lies in aberrant NOTCH4 signaling that deviates endosomal trafficking and minimizes exosome production. By transcriptionally controlling the profile of RAB GTPases, NOTCH4 signaling restricted vesicular secretion of the enzyme NADPH oxidase 2 (NOX2). Specifically, NOTCH4hiCD8+ Treg cells increased RAB5A and RAB11A expression and suppressed RAB7A, culminating in the accumulation of early and recycling endosomes and sequestering of NOX2 in an intracellular compartment. RAB7AloCD8+ Treg cells failed in the surface translocation and exosomal release of NOX2. NOTCH4hiRAB5AhiRAB7AloRAB11AhiCD8+ Treg cells left adaptive immunity unopposed, enabling a breakdown in tissue tolerance and aggressive vessel wall inflammation. Inhibiting NOTCH4 signaling corrected the defect and protected arteries from inflammatory insult. This study implicates NOTCH4-dependent transcriptional control of RAB proteins and intracellular vesicle trafficking in autoimmune disease and in vascular inflammation.

NOX2-Derived Reactive Oxygen Species in Cancer.

The formation of reactive oxygen species (ROS) by the myeloid cell NADPH oxidase NOX2 is critical for the destruction of engulfed microorganisms. However, recent studies imply that ROS, formed by NOX2+ myeloid cells in the malignant microenvironment, exert multiple actions of relevance to the growth and spread of neoplastic cells. By generating ROS, tumor-infiltrating myeloid cells and NOX2+ leukemic myeloid cells may thus (i) compromise the function and viability of adjacent cytotoxic lymphocytes, including natural killer (NK) cells and T cells, (ii) oxidize DNA to trigger cancer-promoting somatic mutations, and (iii) affect the redox balance in cancer cells to control their proliferation and survival. Here, we discuss the impact of NOX2-derived ROS for tumorigenesis, tumor progression, regulation of antitumor immunity, and metastasis. We propose that NOX2 may be a targetable immune checkpoint in cancer.

Different roles of intracellular and extracellular reactive oxygen species of neutrophils in type 2 diabetic mice with invasive aspergillosis.

Diabetic patients have an increased risk of invasive aspergillosis (IA), but the mechanism is still unclear. Reactive oxygen species (ROS) produced by neutrophils play a key role in defense against Aspergillus infection. Since diabetes mellitus affects the production of ROS from neutrophils, the purpose of this study is to investigate whether this effect is related to the susceptibility of diabetic mice to IA. C57BL/6 mice were used to establish type 2 diabetes mellitus (T2DM) model, and IA was induced by airway infection with Aspergillus fumigatus. After infection, the fungal load, neutrophil count and ROS content in the lung tissues of T2DM mice were higher than those in the control mice, and the inflammation of the lung tissue was more serious. After being exposed to hyphae in vitro, compared with the control group, neutrophils in T2DM mice had higher apoptosis rate and intracellular ROS content, as well as lower viability, extracellular ROS content and fungicidal ability. In summary, after T2DM mice are infected with A. fumigatus, the reduction of extracellular ROS produced by neutrophils may lead to a decrease in fungicidal ability, while the increase of intracellular ROS is related to neutrophil and lung tissue damage.

Idelalisib Rescues Natural Killer Cells from Monocyte-Induced Immunosuppression by Inhibiting NOX2-Derived Reactive Oxygen Species.

The phosphatidylinositol-4,5-bisphosphate-3 kinase-δ (PI3Kδ) inhibitor idelalisib, used alone or in combination with anti-CD20, is clinically efficacious in B-cell lymphoma and chronic lymphocytic leukemia (CLL) by promoting apoptosis of malignant B cells. PI3K regulates the formation of reactive oxygen species (ROS) by the myeloid NADPH oxidase NOX2, but the role of PI3Kδ in myeloid cell-induced immunosuppression is unexplored. We assessed the effects of idelalisib on the spontaneous and IgG antibody-induced ROS production by human monocytes, on ROS-induced cell death of human natural killer (NK) cells, and on tumor cell clearance in an NK cell-dependent mouse model of metastasis. Idelalisib potently and efficiently inhibited the formation of NOX2-derived ROS from monocytes and rescued NK cells from ROS-induced cell death. Idelalisib also promoted NK cell cytotoxicity against anti-CD20-coated primary human CLL cells and cultured malignant B cells. Experiments using multiple PI3K inhibitors implicated the PI3Kδ isoform in regulating NOX2-induced ROS formation and immunosuppression. In B6 mice, systemic treatment with idelalisib significantly reduced the formation of lung metastases from intravenously injected melanoma cells but did not affect metastasis in B6.129S6-Cybbtm1Din (Nox2 -/-) mice or in NK cell-deficient mice. Our results imply that idelalisib rescues NK cells from NOX2/ROS-dependent immunosuppression and thus exerts antineoplastic efficacy beyond B-cell inhibition.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Innate Effector Systems in Primary Human Macrophages Sensitize Multidrug-Resistant Klebsiella pneumoniae to Antibiotics.

Infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae are difficult to treat with conventional antibiotics. Thus, alternative strategies to control the growth of MDR Klebsiella are warranted. We hypothesized that activation of innate effector systems could sensitize MDR K. pneumoniae to conventional antibiotics. Thus, human primary macrophages were stimulated with compounds known to activate innate immunity (vitamin D3, phenylbutyrate [PBA], and the aroylated phenylenediamine HO53) and then infected with MDR Klebsiella in the presence or absence of antibiotics. Antibiotics alone were ineffective against MDR Klebsiella in the cellular model, whereas vitamin D3, PBA, and HO53 reduced intracellular growth by up to 70%. The effect was further improved when the innate activators were combined with antibiotics. Vitamin D3- and PBA-induced bacterial killing was dependent on CAMP gene expression, whereas HO53 needed the production of reactive oxygen species (ROS), as shown in cells where the CYBB gene was silenced and in cells from a patient with reduced ROS production due to a deletion in the CYBB gene and skewed lyonization. The combination of innate effector activation by vitamin D3, PBA, and HO53 was effective in sensitizing MDR Klebsiella to conventional antibiotics in a primary human macrophage model. This study provides new evidence for future treatment options for K. pneumoniae.

Elevated expression of toll-like receptor 4 is associated with NADPH oxidase-induced oxidative stress in B cells of children with autism.

Autism spectrum disorder (ASD) is a childhood disorder with neurodevelopmental dysfunction which manifests as impairment in social behavior and communication skills. B cells play an important role in immune dysfunction where toll-like receptor 4 (TLR4) may contribute through oxidative inflammatory process. TLR4 related signaling and oxidative stress have been reported in the periphery of ASD subjects, however it has not been evaluated in peripheral B cells of ASD subjects and compared with typically developing control (TDC) children. This study evaluated TLR4 expression and related signaling [Bruton's tyrosine kinase (BTK), spleen tyrosine kinase (SYK), NF-kB, NADPH oxidase (NOX2), nitrotyrosine, superoxide dismutase (SOD)] in ASD and TDC subjects. Current investigation in B cells shows that ASD subjects have increased TLR4 expression and oxidative stress as exhibited by upregulated NOX2 and nitrotyrosine expression as compared to TDC subjects. B cell relevant pathways, BTK/SYK/NF-kB were also upregulated in B cells of ASD group. Treatment with TLR4 agonist, LPS led to upregulation of NOX2 and nitrotyrosine in B cells of ASD whereas it had no significant effect on TDC subjects. Treatment with NF-kB inhibitor caused inhibition of LPS-induced upregulation of NOX2 and nitrotyrosine in B cells of ASD. Therefore, current investigation proposes the notion that TLR4 expression is elevated in B cells which is associated with increased NF-kB signaling and oxidant stress in ASD subjects. In short, peripheral B cells could contribute to systemic oxidative inflammation and contribute to the immune dysfunction in ASD.

Exophilin-5 regulates allergic airway inflammation by controlling IL-33-mediated Th2 responses.

A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma.

Walnut Polyphenol Extract Protects against Malathion- and Chlorpyrifos-Induced Immunotoxicity by Modulating TLRx-NOX-ROS.

Malathion (MT) and chlorpyrifos (CPF) are immunotoxic organophosphate pesticides that are used extensively in agriculture worldwide. Dietary polyphenols protect against a variety of toxins. In this study, walnut polyphenol extract (WPE) prevents MT- or CPF-induced toxicity to splenic lymphocytes in vitro. WPE promotes the proliferation of MT-exposed splenocytes, as indicated by increases in the proportions of splenic T-lymphocyte subpopulations (CD3+, CD4+, and CD8+ T cells) and levels of T-cell-related cytokines interleukin (IL)-2, interferon-γ, IL-4, and granzyme B, and decreases the apoptosis-associated proteins Bax and p53. WPE also significantly enhances the proliferation of CPF-exposed splenic B lymphocytes (CD19+ B cells) and levels of the B-cell-related cytokine IL-6, leading to decreases of the apoptosis-associated proteins Bax and p53. These effects are related to reduced production of reactive oxygen species (ROS), as evidenced by normalized hydroxyl radical (•OH), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) levels, which are associated with decreased expression of NADPH oxidase 2 (NOX2) and dual oxidase 1 (DUOX1). WPE inhibits the production of ROS and expression of NOX by regulating toll-like receptors 4 and 7 in MT- and CPF-exposed splenic lymphocytes. In conclusion, WPE protects against MT- or CPF-mediated immunotoxicity and inhibits oxidative damage by modulating toll-like receptor (TLR)x-NOX-ROS.

Cancer-Associated Fibroblasts Promote Immunosuppression by Inducing ROS-Generating Monocytic MDSCs in Lung Squamous Cell Carcinoma.

Cancer-associated fibroblasts (CAF) represent a functionally heterogeneous population of activated fibroblasts that constitutes a major component of tumor stroma. Although CAFs have been shown to promote tumor growth and mediate resistance to chemotherapy, the mechanisms by which they may contribute to immune suppression within the tumor microenvironment (TME) in lung squamous cell carcinoma (LSCC) remain largely unexplored. Here, we identified a positive correlation between CAF and monocytic myeloid cell abundances in 501 primary LSCCs by mining The Cancer Genome Atlas data sets. We further validated this finding in an independent cohort using imaging mass cytometry and found a significant spatial interaction between CAFs and monocytic myeloid cells in the TME. To delineate the interplay between CAFs and monocytic myeloid cells, we used chemotaxis assays to show that LSCC patient-derived CAFs promoted recruitment of CCR2+ monocytes via CCL2, which could be reversed by CCR2 inhibition. Using a three-dimensional culture system, we found that CAFs polarized monocytes to adopt a myeloid-derived suppressor cell (MDSC) phenotype, characterized by robust suppression of autologous CD8+ T-cell proliferation and IFNγ production. We further demonstrated that inhibiting IDO1 and NADPH oxidases, NOX2 and NOX4, restored CD8+ T-cell proliferation by reducing reactive oxygen species (ROS) generation in CAF-induced MDSCs. Taken together, our study highlights a pivotal role of CAFs in regulating monocyte recruitment and differentiation and demonstrated that CCR2 inhibition and ROS scavenging abrogate the CAF-MDSC axis, illuminating a potential therapeutic path to reversing the CAF-mediated immunosuppressive microenvironment.

MyD88-dependent influx of monocytes and neutrophils impairs lymph node B cell responses to chikungunya virus infection via Irf5, Nos2 and Nox2.

Humoral immune responses initiate in the lymph node draining the site of viral infection (dLN). Some viruses subvert LN B cell activation; however, our knowledge of viral hindrance of B cell responses of important human pathogens is lacking. Here, we define mechanisms whereby chikungunya virus (CHIKV), a mosquito-transmitted RNA virus that causes outbreaks of acute and chronic arthritis in humans, hinders dLN antiviral B cell responses. Infection of WT mice with pathogenic, but not acutely cleared CHIKV, induced MyD88-dependent recruitment of monocytes and neutrophils to the dLN. Blocking this influx improved lymphocyte accumulation, dLN organization, and CHIKV-specific B cell responses. Both inducible nitric oxide synthase (iNOS) and the phagocyte NADPH oxidase (Nox2) contributed to impaired dLN organization and function. Infiltrating monocytes expressed iNOS through a local IRF5- and IFNAR1-dependent pathway that was partially TLR7-dependent. Together, our data suggest that pathogenic CHIKV triggers the influx and activation of monocytes and neutrophils in the dLN that impairs virus-specific B cell responses.

Does Dysregulation of Redox State Underpin the Decline of Innate Immunity with Aging?

Significance: Antibacterial defense invokes the innate immune system as a first responder, with neutrophils phagocytozing and forming neutrophil extracellular traps around pathogens in a reactive oxygen species (ROS)-dependent manner. Increased NOX2 activity and mitochondrial ROS production in phagocytic, antigen-presenting cells (APCs) affect local cytokine secretion and proteolysis of antigens for presentation to T cells at the immune synapse. Uncontrolled oxidative post-translational modifications to surface and cytoplasmic proteins in APCs during aging can impair innate immunity. Recent Advances: NOX2 plays a role in the maturation of dendritic cells, but paradoxically NOX2 activity has also been shown to promote viral pathogenicity. Accumulating evidence suggests that a reducing environment is essential to inhibit pathogen proliferation, facilitate antigenic processing in the endosomal lumen, and enable an effective immune synapse between APCs and T cells. This suggests that the kinetics and location of ROS production and reducing potential are important for effective innate immunity. Critical Issues: During aging, innate immune cells are less well able to phagocytoze, kill bacteria/viruses, and process proteins into antigenic peptides-three key steps that are necessary for developing a specific targeted response to protect against future exposure. Aberrant control of ROS production and impaired Nrf2-dependent reducing potential may contribute to age-associated immune decline. Future Directions: Local changes in redox potential may be achieved through adjuvant formulations to improve innate immunity. Further work is needed to understand the timing of delivery for redox modulators to facilitate innate immune cell recruitment, survival, antigen processing and presentation activity without disrupting essential ROS-dependent bacterial killing.