Reactive Oxygen Species Production In Macrophages Research Articles

Forsythia velutina Nakai extract: A promising therapeutic option for atopic dermatitis through multiple cell type modulation.

Atopic dermatitis (AD) is a complex condition characterized by impaired epithelial barriers and dysregulated immune cells. In this study, we demonstrated Forsythia velutina Nakai extract (FVE) simultaneously inhibits basophils, macrophages, keratinocytes, and T cells that are closely interrelated in AD development. We analyzed the effect of FVE on nitric oxide and reactive oxygen species (ROS) production in macrophages, basophil degranulation, T cell activation, and tight junctions in damaged keratinocytes. Expression of cell-type-specific inflammatory mediators was analyzed, and the underlying signaling pathways for anti-inflammatory effects of FVE were investigated. The anti-inflammatory effects of FVE were validated using a DNCB-induced mouse model of AD. Anti-inflammatory activity of compounds isolated from FVE was validated in each immune cell type. FVE downregulated the expression of inflammatory mediators and ROS production in macrophages through TLR4 and NRF2 pathways modulation. It significantly reduced basophil degranulation and expression of type 2 (T2) and pro-inflammatory cytokines by perturbing FcεRI signaling. Forsythia velutina Nakai extract also robustly inhibited the expression of T2 cytokines in activated T cells. Furthermore, FVE upregulated the expression of tight junction molecules in damaged keratinocytes and downregulated leukocyte attractants, as well as IL-33, an inducer of T2 inflammation. In the AD mouse model, FVE showed superior improvement in inflammatory cell infiltration and skin structure integrity compared to dexamethasone. Dimatairesinol, a lignan dimer, was identified as the most potent anti-inflammatory FVE compound. Forsythia velutina Nakai extract and its constituent compounds demonstrate promising efficacy as a therapeutic option for prolonged AD treatment by independently inhibiting various cell types associated with AD and disrupting the deleterious link between them.

Quercetin Reprograms Immunometabolism of Macrophages via the SIRT1/PGC-1α Signaling Pathway to Ameliorate Lipopolysaccharide-Induced Oxidative Damage.

The redox system is closely related to changes in cellular metabolism. Regulating immune cell metabolism and preventing abnormal activation by adding antioxidants may become an effective treatment for oxidative stress and inflammation-related diseases. Quercetin is a naturally sourced flavonoid with anti-inflammatory and antioxidant activities. However, whether quercetin can inhibit LPS-induced oxidative stress in inflammatory macrophages by affecting immunometabolism has been rarely reported. Therefore, the present study combined cell biology and molecular biology methods to investigate the antioxidant effect and mechanism of quercetin in LPS-induced inflammatory macrophages at the RNA and protein levels. Firstly, quercetin was found to attenuate the effect of LPS on macrophage proliferation and reduce LPS-induced cell proliferation and pseudopodia formation by inhibiting cell differentiation, as measured by cell activity and proliferation. Subsequently, through the detection of intracellular reactive oxygen species (ROS) levels, mRNA expression of pro-inflammatory factors and antioxidant enzyme activity, it was found that quercetin can improve the antioxidant enzyme activity of inflammatory macrophages and inhibit their ROS production and overexpression of inflammatory factors. In addition, the results of mitochondrial morphology and mitochondrial function assays showed that quercetin could upregulate the mitochondrial membrane potential, ATP production and ATP synthase content decrease induced by LPS, and reverse the mitochondrial morphology damage to a certain extent. Finally, Western blotting analysis demonstrated that quercetin significantly upregulated the protein expressions of SIRT1 and PGC-1α, that were inhibited by LPS. And the inhibitory effects of quercetin on LPS-induced ROS production in macrophages and the protective effects on mitochondrial morphology and membrane potential were significantly decreased by the addition of SIRT1 inhibitors. These results suggested that quercetin reprograms the mitochondria metabolism of macrophages through the SIRT1/PGC-1α signaling pathway, thereby exerting its effect of alleviating LPS-induced oxidative stress damage.

Diallyl trisulfide inhibits monosodium urate-induced NLRP3 inflammasome activation via NOX3/4-dependent mitochondrial oxidative stress in RAW 264.7 and bone marrow-derived macrophages

BackgroundMonosodium urate (MSU) crystals are associated with gouty inflammatory diseases. MSU-associated inflammation is majorly triggered by NOD-like receptor protein 3 (NLRP3) inflammasome that promotes interleukin (IL)-1β secretion. Although diallyl trisulfide (DATS) is well-known polysulfide garlic compounds with anti-inflammatory effects, its action in MSU-induced inflammasome activation has not been known yet. PurposeThe objective of the current study was to investigate anti-inflammasome effects and mechanisms of DATS in RAW 264.7 and bone marrow-derived macrophages (BMDM). MethodsThe concentrations of IL-1β were analyzed with enzyme-linked immunosorbent assay. The MSU-induced mitochondrial damage and reactive oxygen species (ROS) production were detected by fluorescence microscope and flow cytometry. The protein expressions of NLRP3 signaling molecules, NADPH oxidase (NOX) 3/4 were assessed with Western blotting. ResultsDATS suppressed MSU-induced IL-1β and caspase-1 accompanied by decreased inflammasome complex formation in RAW 264.7 and BMDM. In addition, DATS restored mitochondrial damage. DATS downregulated NOX 3/4 that were upregulated by MSU as predicted by gene microarray and confirmed by Western blotting. ConclusionThis study first reports mechanistic finding that DATS alleviates MSU-induced NLRP3 inflammasome by mediating NOX3/4-dependent mitochondrial ROS production in macrophages in vitro and ex vivo, suggesting DATS could be effective therapeutic candidate for gouty inflammatory condition.

Protein phosphatase 2A regulates xanthine oxidase-derived ROS production in macrophages and influx of inflammatory monocytes in a murine gout model.

Background: Gout is a common arthritis, due to deposition of monosodium urate (MSU) crystals which results in IL-1β secretion by tissue-resident macrophages. Xanthine oxidase (XO) catalyzes uric acid (UA) production and in the process, reactive oxygen species (ROS) are generated which contributes to NLRP3 inflammasome activation. Protein phosphatase 2A (PP2A) may be involved in regulating inflammatory pathways in macrophages. The objective of this study was to investigate whether PP2A regulates gout inflammation, mediated by XO activity modulation. We studied UA and ROS generations in MSU stimulated murine bone marrow derived macrophages (BMDMs) in response to fingolimod phosphate, a PP2A activator, and compared its anti-inflammatory efficacy to that of an XO inhibitor, febuxostat. Methods: BMDMs were stimulated with MSU, GM-CSF/IL-1β or nigericin ± fingolimod (2.5μM) or febuxostat (200μM) and UA levels, ROS, XO, and PP2A activities, Xdh (XO) expression and secreted IL-1β levels were determined. PP2A activity and IL-1β in MSU stimulated BMDMs ± N-acetylcysteine (NAC) (10μM) ± okadaic acid (a PP2A inhibitor) were also determined. M1 polarization of BMDMs in response to MSU ± fingolimod treatment was assessed by a combination of iNOS expression and multiplex cytokine assay. The in vivo efficacy of fingolimod was assessed in a murine peritoneal model of acute gout where peritoneal lavages were studied for pro-inflammatory classical monocytes (CMs), anti-inflammatory nonclassical monocytes (NCMs) and neutrophils by flow cytometry and IL-1β by ELISA. Results: Fingolimod reduced intracellular and secreted UA levels (p < 0.05), Xdh expression (p < 0.001), XO activity (p < 0.001), ROS generation (p < 0.0001) and IL-1β secretion (p < 0.0001), whereas febuxostat enhanced PP2A activity (p < 0.05). NAC treatment enhanced PP2A activity and reduced XO activity and PP2A restoration mediated NAC's efficacy as co-treatment with okadaic acid increased IL-1β secretion (p < 0.05). Nigericin activated caspase-1 and reduced PP2A activity (p < 0.001) and fingolimod reduced caspase-1 activity in BMDMs (p < 0.001). Fingolimod reduced iNOS expression (p < 0.0001) and secretion of IL-6 and TNF-α (p < 0.05). Fingolimod reduced CMs (p < 0.0001), neutrophil (p < 0.001) and IL-1β (p < 0.05) lavage levels while increasing NCMs (p < 0.001). Conclusion: Macrophage PP2A is inactivated in acute gout by ROS and a PP2A activator exhibited a broad anti-inflammatory effect in acute gout in vitro and in vivo.

Receptor for Advanced Glycation End-Products Promotes Activation of Alveolar Macrophages through the NLRP3 Inflammasome/TXNIP Axis in Acute Lung Injury.

The roles of thioredoxin-interacting protein (TXNIP) and receptor for advanced glycation end-products (RAGE)-dependent mechanisms of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome-driven macrophage activation during acute lung injury are underinvestigated. Cultured THP-1 macrophages were treated with a RAGE agonist (S100A12), with or without a RAGE antagonist; cytokine release and intracytoplasmic production of reactive oxygen species (ROS) were assessed in response to small interfering RNA knockdowns of TXNIP and NLRP3. Lung expressions of TXNIP and NLRP3 and alveolar levels of IL-1β and S100A12 were measured in mice after acid-induced lung injury, with or without administration of RAGE inhibitors. Alveolar macrophages from patients with acute respiratory distress syndrome and from mechanically ventilated controls were analyzed using fluorescence-activated cell sorting. In vitro, RAGE promoted cytokine release and ROS production in macrophages and upregulated NLRP3 and TXNIP mRNA expression in response to S100A12. TXNIP inhibition downregulated NLRP3 gene expression and RAGE-mediated release of IL-1β by macrophages in vitro. In vivo, RAGE, NLRP3 and TXNIP lung expressions were upregulated during experimental acute lung injury, a phenomenon being reversed by RAGE inhibition. The numbers of cells expressing RAGE, NLRP3 and TXNIP among a specific subpopulation of CD16+CD14+CD206- (“pro-inflammatory”) alveolar macrophages were higher in patients with lung injury. This study provides a novel proof-of-concept of complex RAGE–TXNIP–NLRP3 interactions during macrophage activation in acute lung injury.

Mycobacterium tuberculosis whiB3 and Lipid Metabolism Genes Are Regulated by Host Induced Oxidative Stress

The physiological state of the human macrophage may impact the metabolism and the persistence of Mycobacterium tuberculosis. This pathogen senses and counters the levels of O2, CO, reactive oxygen species (ROS), and pH in macrophages. M. tuberculosis responds to oxidative stress through WhiB3. The goal was to determine the effect of NADPH oxidase (NOX) modulation and oxidative agents on the expression of whiB3 and genes involved in lipid metabolism (lip-Y, Icl-1, and tgs-1) in intracellular mycobacteria. Human macrophages were first treated with NOX modulators such as DPI (ROS inhibitor) and PMA (ROS activator), or with oxidative agents (H2O2 and generator system O2•−), and then infected with mycobacteria. We determined ROS production, cell viability, and expression of whiB3, as well as genes involved in lipid metabolism. PMA, H2O2, and O2•− increased ROS production in human macrophages, generating oxidative stress in bacteria and augmented the gene expression of whiB3, lip-Y, Icl-1, and tgs-1. Our results suggest that ROS production in macrophages induces oxidative stress in intracellular bacteria inducing whiB3 expression. This factor may activate the synthesis of reserve lipids produced to survive in the latency state, which allows its persistence for long periods within the host.

Vitamin D Receptor Genetic Polymorphisms Associate With a Decreased Susceptibility to Extremity Osteomyelitis Partly by Inhibiting Macrophage Apoptosis Through Inhibition of Excessive ROS Production via VDR-Bmi1 Signaling.

Background: Previous studies had reported that vitamin D receptor (VDR) gene polymorphisms were related to the development of several inflammatory disorders. However, potential links between such variations and the risk of developing a bone infection and underlying mechanisms remain unclear. This study aimed to analyze potential associations between VDR genetic variations and susceptibility to extremity osteomyelitis (OM) in a Chinese Han population and investigate potential mechanisms. Methods: Between January 2016 and August 2020, altogether 398 OM patients and 368 healthy controls were genotyped for six VDR gene polymorphisms, including ApaI (rs7975232), BsmI (rs1544410), FokI (rs2228570), TaqI (rs731236), GATA (rs4516035), and Cdx-2 (rs11568820) by the SNaPshot genotyping method. Then, male C57BL/6 mice were randomly divided into vitamin D–standard, –excess, –deficient, and –rescued groups. One week after making the model surgery, OM occurrence and severity were assessed using the bacterial count and histopathological staining. In vitro, phagocytosis, apoptosis, and bactericidal ability of macrophages were evaluated by overexpression or knockdown of VDR protein. Results: Significant associations were found among rs7975232, rs1544410, and OM development by the recessive model (AA vs. AC + CC, p = 0.037, OR = 0.594), homozygous model (AA vs. CC, p = 0.033, OR = 0.575), and heterozygous model (CT vs. CC, p = 0.049, OR = 0.610), respectively. Patients with the AA genotype of rs7975232 had a relatively higher mean level of vitamin D than those with AC and CC genotypes (22.5 vs. 20.7 vs. 19.0 ng/ml). Similarly, patients with CT genotype of rs1544410 had a relatively higher mean vitamin D level than those with CC genotype (20.94 vs. 19.89 ng/ml). Outcomes of in vivo experiments showed that the femoral bacterial load of vitamin D–deficient mice was highest among different vitamin D dose groups, with the most severe histopathological features of infection, and vitamin D supplementation partly reversed the changes. While in vitro experiment results revealed that active vitamin D promoted phagocytosis and sterilization of macrophages and inhibited apoptosis during infection. Reactive oxygen species (ROS) inhibitor inhibited apoptosis of macrophages induced by bacterial infection. Active vitamin D inhibited excessive ROS production in macrophages via the VDR-Bmi1 signaling pathway. Conclusion: In this Chinese cohort, ApaI and BsmI are associated with a decreased risk of OM development by influencing serological vitamin D level, the latter of which reduced macrophage apoptosis with inhibition of excessive ROS production via the VDR-Bmi1 signaling pathway.

ROS-responsive liposomes as an inhaled drug delivery nanoplatform for idiopathic pulmonary fibrosis treatment via Nrf2 signaling.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease with pathophysiological characteristics of transforming growth factor-β (TGF-β), and reactive oxygen species (ROS)-induced excessive fibroblast-to-myofibroblast transition and extracellular matrix deposition. Macrophages are closely involved in the development of fibrosis. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a key molecule regulating ROS and TGF-β expression. Therefore, Nrf2 signaling modulation might be a promising therapy for fibrosis. The inhalation-based drug delivery can reduce systemic side effects and improve therapeutic effects, and is currently receiving increasing attention, but direct inhaled drugs are easily cleared and difficult to exert their efficacy. Therefore, we aimed to design a ROS-responsive liposome for the Nrf2 agonist dimethyl fumarate (DMF) delivery in the fibrotic lung. Moreover, we explored its therapeutic effect on pulmonary fibrosis and macrophage activation.ResultsWe synthesized DMF-loaded ROS-responsive DSPE-TK-PEG@DMF liposomes (DTP@DMF NPs). DTP@DMF NPs had suitable size and negative zeta potential and excellent capability to rapidly release DMF in a high-ROS environment. We found that macrophage accumulation and polarization were closely related to fibrosis development, while DTP@DMF NPs could attenuate macrophage activity and fibrosis in mice. RAW264.7 and NIH-3T3 cells coculture revealed that DTP@DMF NPs could promote Nrf2 and downstream heme oxygenase-1 (HO-1) expression and suppress TGF-β and ROS production in macrophages, thereby reducing fibroblast-to-myofibroblast transition and collagen production by NIH-3T3 cells. In vivo experiments confirmed the above findings. Compared with direct DMF instillation, DTP@DMF NPs treatment presented enhanced antifibrotic effect. DTP@DMF NPs also had a prolonged residence time in the lung as well as excellent biocompatibility.ConclusionsDTP@DMF NPs can reduce macrophage-mediated fibroblast-to-myofibroblast transition and extracellular matrix deposition to attenuate lung fibrosis by upregulating Nrf2 signaling. This ROS-responsive liposome is clinically promising as an ideal delivery system for inhaled drug delivery.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s12951-022-01435-4.

Epoxyeicosatrienoic acids inhibit the activation of NLRP3 inflammasome in murine macrophages.

Epoxyeicosatrienoic acids (EETs) derived from arachidonic acid exert anti-inflammation effects. We have reported that blocking the degradation of EETs with a soluble epoxide hydrolase (sEH) inhibitor protects mice from lipopolysaccharide (LPS)-induced acute lung injury (ALI). The underlying mechanisms remain essential questions. In this study, we investigated the effects of EETs on the activation of nucleotide-binding domain leucine-rich repeat-containing receptor, pyrin domain-containing-3 (NLRP3) inflammasome in murine macrophages. In an LPS-induced ALI murine model, we found that sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl), TPPU, profoundly attenuated the pathological injury and inhibited the activation of the NLRP3 inflammasome, characterized by the reduction of the protein expression of NLRP3, ASC, pro-caspase-1, interleukin precursor (pro-IL-1β), and IL-1β p17 in the lungs of LPS-treated mice. In vitro, primary peritoneal macrophages from C57BL/6 were primed with LPS and activated with exogenous adenosine triphosphate (ATP). TPPU treatment remarkably reduced the expression of NLRP3 inflammasome-related molecules and blocked the activation of NLRP3 inflammasome. Importantly, four EETs (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) inhibited the activation of NLRP3 inflammasome induced by LPS + ATP or LPS + nigericin in macrophages in various degree. While the inhibitory effect of 5,6-EET was the weakest. Mechanismly, EETs profoundly decreased the content of reactive oxygen species (ROS) and restored the calcium overload in macrophages receiving LPS + ATP stimulation. In conclusion, this study suggests that EETs inhibit the activation of the NLRP3 inflammasome by suppressing calcium overload and ROS production in macrophages, contributing to the therapeutic potency to ALI.

Type I Interferons Ameliorate Zinc Intoxication of Candida glabrata by Macrophages and Promote Fungal Immune Evasion.

SummaryHost and fungal pathogens compete for metal ion acquisition during infectious processes, but molecular mechanisms remain largely unknown. Here, we show that type I interferons (IFNs-I) dysregulate zinc homeostasis in macrophages, which employ metallothionein-mediated zinc intoxication of pathogens as fungicidal response. However, Candida glabrata can escape immune surveillance by sequestering zinc into vacuoles. Interestingly, zinc-loading is inhibited by IFNs-I, because a Janus kinase 1 (JAK1)-dependent suppression of zinc homeostasis affects zinc distribution in macrophages as well as generation of reactive oxygen species (ROS). In addition, systemic fungal infections elicit IFN-I responses that suppress splenic zinc homeostasis, thereby altering macrophage zinc pools that otherwise exert fungicidal actions. Thus, IFN-I signaling inadvertently increases fungal fitness both in vitro and in vivo during fungal infections. Our data reveal an as yet unrecognized role for zinc intoxication in antifungal immunity and suggest that interfering with host zinc homeostasis may offer therapeutic options to treat invasive fungal infections.

Role of Aggregatibacter actinomycetemcomitans-induced autophagy in inflammatory response.

Aggressive periodontitis is characterized by the early-onset and rapid progression of periodontal destruction and is closely associated with Aggregatibacter actinomycetemcomitans. Autophagy is a conserved process that is critical for removing damaged proteins, organelles, and even intracellular pathogens. Therefore, this study examined whether A. actinomycetemcomitans induces autophagy. In addition, the relationship among autophagy, bacterial internalization, and inflammatory molecules in periodontal aggressive inflammation was analyzed. The expression of autophagy-related proteins in human gingival tissue and THP-1 cells was assessed by Western blot analysis. The formation of light chain 3 (LC3) puncta was examined by confocal microscopy. The degree of bacterial internalization into the cells was determined by the viable cell count. Phagocytosis and reactive oxygen species (ROS) production were measured using confocal microscopy and flow cytometry. When macrophages were infected with live A. actinomycetemcomitans, the autophagy influx was activated by the increase in LC3-II, autophagy-related gene 5/12, and Beclin-1 expression through the Toll-like receptors and extracellular signal-regulated kinase signaling pathways. The inhibition of A. actinomycetemcomitans-induced autophagy suppressed bacterial internalization via phagocytosis into the macrophages and increased interleukin (IL)-1β production. Moreover, treatment with an ROS inhibitor inhibited these enhanced inflammatory responses. A. actinomycetemcomitans-induced autophagy promotes bacterial internalization by phagocytosis, which restricts the excessive inflammatory response by downregulating IL-1β and ROS production in macrophages. Thus, A. actinomycetemcomitans-induced autophagy and its role in regulating the inflammatory response may play an important role in the aggressive periodontal inflammatory process, and be a target for the development of new periodontal therapies.

Xanthoplanine attenuates macrophage polarization towards M1 and inflammation response via disruption of CrkL-STAT5 complex

Monocyte infiltration and macrophage polarization are widely considered as pivotal steps for the initiation and progression of atherosclerosis. Previous studies suggested that zanthoxylum piperitum had strong analgesic and anti-inflammatory effects. However, it remains unclear whether zanthoxylum piperitum inhibits inflammation via macrophage function. In the present study, we investigated the effects of xanthoplanine (the total alkaloid extract of zanthoxylum piperitum) on macrophage function. CCK-8 kit was performed to determine cell viability and the preferred concentration of xanthoplanine. We assayed the effects of xanthoplanine on markers of macrophage polarization and inflammation via quantitative PCR and enzyme-linked immunosorbent assay, and measured the production of reactive oxygen species (ROS) by flow cytometry. Immunoblots, co-immunoprecipitation, immunofluorescence and Luciferase activity were performed to investigate the molecular mechanism of STAT signaling pathway in response to xanthoplanine. We found that xanthoplanine (50 and 100 μM) significantly reduced M1 polarization and promoted M2 polarization. The contents of inflammatory cytokines measured by ELISA were markedly decreased in macrophages pretreated with xanthoplanine, compared with those induced by LPS and IFN-γ. In parallel, xanthoplanine alleviated the production of ROS in macrophages induced by LPS and IFN-γ. Moreover, xanthoplanine alleviated STAT5 phosphorylation and blocked STAT5 nuclear translocation without alterations in CrkL expression, subsequently interrupting the interaction between p-STAT5 and CrkL. Likewise, xanthoplanine prominently attenuated the transcription activity of STAT5 induced by LPS and IFN-γ but did not affect the transcription activity of STAT1 and STAT3. Xanthoplanine attenuated M1 phenotypic switch and macrophage inflammation via blocking the formation of CrkL-STAT5 complex.

Carbon nanotubes and crystalline silica stimulate robust ROS production, inflammasome activation, and IL-1β secretion in macrophages to induce myofibroblast transformation.

Pulmonary exposure to inhaled particulates elicits complex inflammatory and fibrotic responses that may progress to chronic fibrosis. The fibrogenic potentials of respirable particulates are influenced by their physicochemical properties and their interactions with major pathways to drive fibrotic development in the lung. Macrophages were exposed to six carbon nanotubes (CNTs) of varying dimensions, crystalline silica, or carbon black (CB), with lipopolysaccharide (LPS) and transforming growth factor (TGF)-β1 as positive controls. Macrophage-conditioned media was collected and applied to cultures of human pulmonary fibroblast line WI38-VA13 to induce myofibroblast transformation. Multi-walled and single-walled CNTs (MWCNTs and SWCNTs, respectively) and silica, but not CB, stimulated robust myofibroblast transformation through macrophage-conditioned media. On an equal weight basis, MWCNTs induced higher induction than SWCNTs. High induction was observed for MWCNTs with a long and slender or a short and rigid shape, and silica, atlevelscomparable to those by LPS and TGF-β1. Fibrogenic particulates induced high levels of IL-1β mRNA expression and protein secretion that are required for macrophage-guided myofibroblast transformation. Induction of IL-1β is dependent on the activation of the NLRP3 (NOD-like receptor family, pyrin domain containing 3) inflammasome and ROS (reactive oxygen species) production in macrophages, as inhibition of NLRP3 by MCC950 and reduction of ROS production by N-acetylcysteine blocked NLRP3 activation,IL-1β induction, and fibroblast activation anddifferentiation. Therefore, fibrogenic CNTs and silica, but not CB, elicit robust macrophage-guided myofibroblast transformation, which depends on the induction of IL-1β through the NLRP3 inflammasome pathway andthe increased production of ROS in macrophages.

Novel Formulated Zinc Oxide Nanoparticles Reduce Hwp1 Gene Expression Involved in Biofilm Formation in Candida albicans with Minimum Cytotoxicity Effect on Human Cells

Background: Drug resistance in Candida species, has emerged as a major problem in the public health system worldwide. Application of nanoparticles is proposed as a novel and potential agent for reduction of drug resistance burdens. Objectives: The current study was conducted to evaluate the effects of zinc oxide nanoparticles (ZnO NPs containing chitosan and linoleic acid) on hyphae cell wall proteins (Hwp1) gene expression, a crucial gene in pathogenicity of Candida albicans, and cytotoxicity on human hepatocyte carcinoma (HepG2) cells as well as the production of Reactive Oxygen Species (ROS). Methods: The effects of novel ZnO NPs on expression of Hwp1 gene of C. albicans was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) in comparison to fluconazole as a standard drug. Reactive Oxygen species production was examined in macrophages treated with ZnO NPs relative to non-treated cells. Also, the cytotoxicity effects of ZnO NPs were assessed using the MTT assay against HepG2 cell line. Results: The findings indicated that ZnO NPs significantly decreased the level of Hwp1 gene expression in standard and clinical isolates of C. albicans. Increased level of ROS production in macrophages was found in the presence of ZnO NPs in concentration-dependent manner compared to the control group without exposure of ZnO NPs (P = 0.001). Furthermore, ZnO NPs did not show cytotoxicity activity on HepG2 cells at different concentrations (P > 0.05). Conclusions: Taken together, the newly synthesized ZnO NPs may be a suitable candidate for inhibition of the critical gene responsible for biofilm dispersion and the control of Candida infection with limited cytotoxicity on human cells. However, more studies are required for support of its effect in vitro and in vivo.

Group A Streptococcus (GAS) Inhibits the Oxidative Burst Response in a Human Macrophage‐like Cell Line By Preventing the Formation of NADPH Oxidase Complex

Group A Streptococcus (GAS) is the cause of millions of infections worldwide each year, many of which progress to severe invasive infections such as sepsis, necrotizing fasciitis, and streptococcal toxic shock syndrome. Cells of the human innate immune system typically initiate the oxidative burst response to form reactive oxygen species (ROS) as a killing mechanism in response to pathogens. Other bacterial species including Lactococcus lactis and Staphylococcus aureus engage defense mechanisms such as catalase or pigments to detoxify these ROS. However, GAS lacks such detoxifying enzymes, and its survival in macrophages suggests its use of an alternative mechanism to avoid ROS‐mediated killing. When we infected THP‐1 macrophages with GAS, infected macrophages displayed significantly decreased peroxide production when compared to infection with S. aureus, L. lactis, and Group B Streptococcus. As the NADPH Oxidase (NOX2) complex is responsible for initiating the oxidative burst response and the generation of ROS such as peroxide, these data suggest that GAS may be impairing the production of ROS by either preventing the assembly of the NOX2 complex or its activation. The NOX2 complex forms on the surface of the phagosomal membrane and consists of two membrane proteins, gp91 and p22, which assemble with three cytosolic proteins, p47, p40, and p67. To assess whether GAS prevents the assembly of the NOX2 complex, we performed cell fractionation to isolate phagosomes of GAS and L. lactis‐infected macrophages. Western blot and mass spectrometry data indicate that NOX2 complex formation is indeed impaired in GAS‐infected cells. We are also currently performing immunoprecipitation experiments using infected THP‐1 cell lysates and antibodies for the p22 membrane subunit to look for NOX2 complex formation. Thus, these data will determine if GAS impairs the production of harmful ROS in macrophages by preventing the initial assembly of the NOX2 complex, ultimately allowing their persistence in an innate immune cell designed to kill them.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Long non-coding RNA Lethe regulates hyperglycemia-induced reactive oxygen species production in macrophages.

Type 2 diabetes mellitus is a complex, systemic metabolic disease characterized by insulin resistance and resulting hyperglycemia, which is associated with impaired wound healing. The clinical complications associated with hyperglycemia are attributed, in part, to the increased production of reactive oxygen species (ROS). Recent studies revealed that long non-coding RNAs (lncRNAs) play important regulatory roles in many biological processes. Specifically, lncRNA Lethe has been described as exhibiting an anti-inflammatory effect by binding to the p65 subunit of NFκB and blocking its binding to DNA and the subsequent activation of downstream genes. We therefore hypothesize that dysregulation of Lethe’s expression plays a role in hyperglycemia-induced ROS production. To test our hypothesis, we treated RAW264.7 macrophages with low glucose (5 mM) or high glucose (25 mM) for 24h. High glucose conditions significantly induced ROS production and NOX2 gene expression in RAW cells, while significantly decreasing Lethe gene expression. Overexpression of Lethe in RAW cells eliminated the increased ROS production induced by high glucose conditions, while also attenuating the upregulation of NOX2 expression. Similar results was found also in non-diabetic and diabetic primary macrophage, bone marrow derived macrophage (BMM). Furthermore, overexpression of Lethe in RAW cells treated with high glucose significantly reduced the translocation of p65-NFkB to the nucleus, which resulted in decreased NOX2 expression and ROS production. Interestingly, these findings are consistent with the decreased Lethe gene expression and increased NOX2 gene expression observed in a mouse model of diabetic wound healing. These findings provide the first evidence that lncRNA Lethe is involved in the regulation of ROS production in macrophages through modulation of NOX2 gene expression via NFκB signaling. Moreover, this is the first report to describe a role of lncRNAs, in particular Lethe, in impaired diabetic wound healing. Further studies are warranted to determine if correction of Lethe expression in diabetic wounds could improve healing.

Neutrophil contributions to the induction and regulation of the acute inflammatory response in teleost fish.

Neutrophils are essential to the acute inflammatory response, where they serve as the first line of defense against infiltrating pathogens. We report that, on receiving the necessary signals, teleost (Carassius auratus) neutrophils leave the hematopoietic kidney, enter into the circulation, and dominate the initial influx of cells into a site of inflammation. Unlike mammals, teleost neutrophils represent <5% of circulating leukocytes during periods of homeostasis. However, this increases to nearly 50% immediately after intraperitoneal challenge with zymosan, identifying a period of neutrophilia that precedes the peak influx of neutrophils into the challenge site at 18 h after injection). We demonstrate that neutrophils at the site of inflammation alter their phenotype throughout the acute inflammatory response, and contribute to both the induction and the resolution of inflammation. However, neutrophils isolated during the proinflammatory phase (18 h after injection) produced robust respiratory burst responses, released inflammation-associated leukotriene B(4), and induced macrophages to increase reactive oxygen species production. In contrast, neutrophils isolated at 48 h after infection (proresolving phase) displayed low levels of reactive oxygen species, released the proresolving lipid mediator lipoxin A(4), and downregulated reactive oxygen species production in macrophages before the initiation of apoptosis. Lipoxin A(4) was a significant contributor to the uptake of apoptotic cells by teleost macrophages and also played a role, at least in part, in the downregulation of macrophage reactive oxygen species production. Our results highlight the contributions of neutrophils to both the promotion and the regulation of teleost fish inflammation and provide added context for the evolution of this hematopoietic lineage.

Glutathione prevents preterm parturition and fetal death by targeting macrophage-induced reactive oxygen species production in the myometrium.

Preterm birth is an inflammatory process resulting from the massive infiltration of innate immune cells and the production of proinflammatory cytokines in the myometrium. However, proinflammatory cytokines, which induce labor in vivo, fail to induce labor-associated features in human myometrial cells (MCs). We thus aimed to investigate if reactive oxygen species (ROS) production could be the missing step between immune cell activation and MC response. Indeed, we found that ROS production is increased in the human preterm laboring myometrium (27% ROS producing cells, respectively, versus 2% in nonlaboring controls), with 90% ROS production in macrophages. Using LPS-stimulated myometrial samples and cell coculture experiments, we demonstrated that ROS production is required for labor onset. Furthermore, we showed that ROS are required first in the NADPH oxidase (NADPHox)-2/NF-κB-dependent macrophage response to inflammatory stimuli but, more importantly, to trigger macrophage-induced MCs transactivation. Remarkably, in a murine model of LPS-induced preterm labor (inducing delivery within 17 hours, with no pup survival), cotreatment with glutathione delayed labor onset up to 94 hours and prevented in utero fetal distress, allowing 46% pups to survive. These results suggest that targeting ROS production with the macrophage-permeable antioxidant glutathione could constitute a promising strategy to prevent preterm birth.

Oregonin reduces lipid accumulation and proinflammatory responses in primary human macrophages

Inflammation in the vascular wall is important for the development of atherosclerosis. We have previously shown that inflammatory macrophages are more abundant in human atherosclerotic lesions than in healthy arteries. Activated macrophages produce reactive oxygen species (ROS) that promote local inflammation in atherosclerotic lesions. Here, we investigated the role of oregonin, a diarylheptanoid, on proinflammatory responses in primary human macrophages and found that oregonin decreased cellular lipid accumulation and proinflammatory cytokine secretion. We also found that oregonin decreased ROS production in macrophages. Additionally, we observed that treatment of lipopolysaccharide-exposed macrophages with oregonin significantly induced the expression of antioxidant-related genes, including Heme oxygenase-1 and NADPH dehydrogenase quinone 1. In summary, we have shown that oregonin reduces lipid accumulation, inflammation and ROS production in primary human macrophages, indicating that oregonin has anti-inflammatory bioactivities.

Macrophage inflammasome mediates hyperhomocysteinemia-aggravated abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a serious vascular disease with high mortality. Our previous study suggested that hyperhomocysteinemia (HHcy) exaggerates the occurrence of AAA. Here, we investigated whether macrophage inflammasome is involved in HHcy-aggravated AAA formation. Two independent HHcy-aggravated AAA models, perivascular calcium phosphate–treated C57BL/6 mice and angiotensin II (Ang II)–infused apolipoprotein E-deficient (ApoE−/−) mice were used. NLPR3, caspase 1, and interleukin-1β (IL-1β) levels were higher in aneurysmal lesions of both HHcy models compared to controls, preferentially in macrophages. Similarly, macrophage inflammasome activation was observed in vitro. Folic acid administration reversed the HHcy-accelerated AAA, with ameliorated activation of inflammasome in the tunica adventitia. Lentiviral silencing of NLRP3 significantly ameliorated HHcy-aggravated AAA formation. We observed increased mitochondrial production of reactive oxygen species (ROS) and energy switch from oxidative phosphorylation to glycolysis with excess Hcy in macrophages. Blocking mitochondrial ROS production in macrophages abolished inflammasome activation. Our study highlights the potential importance of macrophage inflammasome in the pathogenesis and development of HHcy-aggravated AAA.