ROS Production In Macrophages Research Articles

S100A8-Mediated NLRP3 Inflammasome-Dependent Pyroptosis in Macrophages Facilitates Liver Fibrosis Progression.

NLRP3 inflammasome-dependent pyroptosis has been implicated in liver fibrosis progression. However, the definite intrahepatic cell types that undergo pyroptosis and the underlying mechanism as well as the clinical importance remain unclear. Here, augmented levels of pyroptosis-related indicators GSDMD, IL-1β, and IL-18 were verified in both liver fibrosis patients and CCl4-induced fibrotic mouse model. Confocal imaging of NLRP3 with albumin, F4/80 or α-SMA revealed that enhanced NLRP3 was mainly localized to kupffer cells (KCs), indicating that KCs are major cell types that undergo pyroptosis. Targeting pyroptosis by inhibitor MCC950 attenuated the severity and ameliorated liver function in fibrosis models. In addition, elevated S100A8 in liver fibrosis patients was correlated with pyroptosis-related indicators. S100A8 stimulated pyroptotic death of macrophages, which resulted in activation of human hepatic stellate cell line LX-2 cells and increased collagen deposition. Mechanistically, S100A8 activated TLR4/NF-κB signaling and upregulated its target genes NLRP3, pro-IL-1β, and pro-IL-18 expression, and induced reactive oxygen (ROS) abundance to activate NLRP3 inflammasome, finally leading to pyroptotic cell death in macrophages. More importantly, circulating GSDMD had the optimal predicting value for liver fibrosis progression. In conclusion, S100A8-mediated NLRP3 inflammasome-dependent pyroptosis by TLR4/NF-κB activation and ROS production in macrophages facilitates liver fibrosis progression. The identified GSDMD has the potential to be a biomarker for liver fibrosis evaluation.

HDM induces distinct immunometabolic phenotype in macrophages in TLR4-dependent manner

Asthma is one of the most common chronic diseases. In many cases it is preceded by the development of an immune response to allergens such as animal fur, dust, pollens and etc. In human population this disease is heterogeneous, and no selective drugs are available at the moment for some endotypes of asthma. The role of the adaptive immune system in the pathogenesis of asthma was extensively studied, while the role of innate immune cells, in particular myeloid cells, was not sufficiently addressed. Myeloid cells, such as macrophages and dendritic cells, are characterized by high plasticity, heterogenicity and ability to undergo polarization in response to various pathogenic stimuli, including those engaging innate immune receptors. Recently, special attention was drawn to the link between polarization of macrophages and cell metabolism. We hypothesized that immunometabolic reprogramming of myeloid cells, in particular, of macrophages and dendritic cells during sensitization with an allergen may affect further immune response and asthma development. To test this hypothesis, we generated distinct types of myeloid cells in vitro from murine bone marrow and analyzed their immunometabolic profiles upon activation with house dust mite extract (HDM) and its key active components. We found that the combination of lipopolysaccharide (LPS) and beta-glucan is sufficient to upregulate proinflammatory cytokine production as well as respiratory and glycolytic capacity of myeloid cells, comparably to HDM. This specific immunometabolic phenotype was associated with altered mitochondrial morphology and possibly with increased ROS production in macrophages. Moreover, we found that both TNF production and metabolic remodeling of macrophages in response to HDM are TLR4-dependent processes. Altogether, these results expand our understanding of molecular mechanisms underlying asthma induction and pathogenesis and may potentially lead to new therapeutic strategies for the treatment of this disease.

Back Cover: Myo‐Inositol in Fermented Sugar Matrix Improves Human Macrophage Function

Mol. Nutr. Food Res. 2022, 66, 202100852 DOI: 10.1002/mnfr.202100852 Fermented Papaya Preparation (FPP) treatment increases macrophage intracellular myo-inositol. FPP and myo-inositol bolsters human macrophage ROS production and phagocytosis. Myo-inositol transporters SMIT1and HMIT are involved in FPP and myo-inositol induced macrophage functions. Myo-inositol improves macrophage functions via ISYNA1 and RAC2-mediated pathway. This is reported by Sashwati Roy and co-workers in article number 2100852.

Macrophage interferon regulatory factor 4 deletion ameliorates aristolochic acid nephropathy via reduced migration and increased apoptosis.

Aristolochic acid (AA) is the causative nephrotoxic alkaloid in AA nephropathy, which results in a tubulointerstitial fibrosis. AA causes direct proximal tubule damage as well as an influx of macrophages, although the role of macrophages in pathogenesis is poorly understood. Here, we demonstrate that AA directly stimulates migration, inflammation, and ROS production in macrophages ex vivo. Cells lacking interferon regulatory factor 4 (IRF4), a known regulator of macrophage migration and phenotype, had a reduced migratory response, though effects on ROS production and inflammation were preserved or increased relative to WT cells. Macrophage-specific IRF4-knockout mice were protected from both acute and chronic kidney effects of AA administration based on functional and histological analysis. Renal macrophages from kidneys of AA-treated macrophage-specific IRF4-knockout mice demonstrated increased apoptosis and ROS production compared with WT controls, indicating that AA directly polarizes macrophages to a promigratory and proinflammatory phenotype. However, knockout mice had reduced renal macrophage abundance following AA administration. While macrophages lacking IRF4 can adopt a proinflammatory phenotype upon AA exposure, their inability to migrate to the kidney and increased rates of apoptosis upon infiltration provide protection from AA in vivo. These results provide evidence of direct AA effects on macrophages in AA nephropathy and add to the growing body of evidence that supports a key role of IRF4 in modulating macrophage function in kidney injury.

The immune response of a warm water fish orange-spotted grouper (Epinephelus coioides) infected with a typical cold water bacterial pathogen Aeromonas salmonicida is AhR dependent

The present study reported the first pathogenic Aeromonas salmonicida (SRW-OG1) isolated from the warm water fish orange-spotted grouper (Epinephelus coioides), and investigated the function of Aryl hydrocarbon receptor (AhR), a ligand-dependent transcriptional factor which has been recently found to be closely associated with immune response in mammals and E. coioides. Our results showed that AhR was activated by an unknown ligand in the spleen, intestine and macrophages. Meanwhile, ahr1a and ahr1b were significantly increased in the spleen, intestine and macrophages, whereas ahr2 was only increased in the intestine, which indicated that the contribution of AhR2 to the immune response may be less than that of AhR1a and AhR1b. Some key genes involved in the macrophage inflammatory response, bacterial recognition, and intestinal immunity were significantly up-regulated in the SRW-OG1 infected E. coioides. Nevertheless, declining macrophage ROS production and down-regulation of related genes were also observed, suggesting that SRW-OG1 utilized its virulence mechanisms to prevent macrophage ROS production. Furthermore, AhR inhibitor 3′, 4′-DMF and the silence of ahr1a or ahr1b significantly rescued the increased IL-1β and IL-8 induced by SRW-OG1 infection, which proved that the induction of IL-1β and IL-8 in E. coioides macrophages was mediated by AhR. However, BPI/LBP, ROS production and related genes were not affected by AhR. The survival rate and immune escape rate of SRW-OG1 in the ahr1a/ahr1b knocked-down and 3′, 4′-DMF treated macrophages were significantly increased compared with those in wild type macrophages. Taken together, it was preliminarily confirmed that ahr1a and ahr1b played an important role in the immune response against A. salmonicida SRW-OG1.

Comparative RNA-Seq transcriptome analyses reveal dynamic time-dependent effects of 56Fe, 16O, and 28Si irradiation on the induction of murine hepatocellular carcinoma

BackgroundOne of the health risks posed to astronauts during deep space flights is exposure to high charge, high-energy (HZE) ions (Z > 13), which can lead to the induction of hepatocellular carcinoma (HCC). However, little is known on the molecular mechanisms of HZE irradiation-induced HCC.ResultsWe performed comparative RNA-Seq transcriptomic analyses to assess the carcinogenic effects of 600 MeV/n 56Fe (0.2 Gy), 1 GeV/n 16O (0.2 Gy), and 350 MeV/n 28Si (0.2 Gy) ions in a mouse model for irradiation-induced HCC. C3H/HeNCrl mice were subjected to total body irradiation to simulate space environment HZE-irradiation, and liver tissues were extracted at five different time points post-irradiation to investigate the time-dependent carcinogenic response at the transcriptomic level. Our data demonstrated a clear difference in the biological effects of these HZE ions, particularly immunological, such as Acute Phase Response Signaling, B Cell Receptor Signaling, IL-8 Signaling, and ROS Production in Macrophages. Also seen in this study were novel unannotated transcripts that were significantly affected by HZE. To investigate the biological functions of these novel transcripts, we used a machine learning technique known as self-organizing maps (SOMs) to characterize the transcriptome expression profiles of 60 samples (45 HZE-irradiated, 15 non-irradiated control) from liver tissues. A handful of localized modules in the maps emerged as groups of co-regulated and co-expressed transcripts. The functional context of these modules was discovered using overrepresentation analysis. We found that these spots typically contained enriched populations of transcripts related to specific immunological molecular processes (e.g., Acute Phase Response Signaling, B Cell Receptor Signaling, IL-3 Signaling), and RNA Transcription/Expression.ConclusionsA large number of transcripts were found differentially expressed post-HZE irradiation. These results provide valuable information for uncovering the differences in molecular mechanisms underlying HZE specific induced HCC carcinogenesis. Additionally, a handful of novel differentially expressed unannotated transcripts were discovered for each HZE ion. Taken together, these findings may provide a better understanding of biological mechanisms underlying risks for HCC after HZE irradiation and may also have important implications for the discovery of potential countermeasures against and identification of biomarkers for HZE-induced HCC.

Antidiabetic potential of Bauhinia forficata Link leaves: a non-cytotoxic source of lipase and glycoside hydrolases inhibitors and molecules with antioxidant and antiglycation properties

Bauhinia forficata Link., a cerrado native plant, is used as a complementary treatment for Type 2 Diabetes Mellitus (T2DM). Several studies involving this plant have shown that it has prominent potential to combat hyperglycemia and oxidative stress. Our objective was suggest the phytochemical constitution of fractions of ethanol extract of B. forficata leaves using HPLC-ESI-MS/MS, and evaluates their activities in enzymatic assays to evaluate their inhibitory potential against α-amylase, α-glucosidase and lipase, as well as their antioxidant and anti-glycation capacities. In addition, we evaluated the cytotoxic effects of these fractions using rodents macrophages and erythrocytes. The ETOAC e ButOH fractions showed high polyphenols concentrations, having been determined 11 flavonoids, including the kaempferitrin, the phytomarker of B. forficata Link. In addition, all fractions presented higher antioxidant and antiglycation activities and prominent capacities to digestive enzymes inhibition. On the other hand, in the cellular assays, none fractions showed cytotoxic and hemolytic effects, able to combat the ROS production in macrophages. Thus, this study presented new results on the biological activities of this plant, contributing to the understanding of the action and effectiveness of its use in the management of diabetes mellitus and its complications.

Transient receptor potential vanilloid 4 is a critical mediator in LPS mediated inflammation by mediating calcineurin/NFATc3 signaling

Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is thought to be an essential component of inflammatory response. However, its role and mechanism in regulating acute lung injury (ALI) and macrophages activation are not well characterized. In our study, we observe that blockade of TRPV4 using GSK2193874 or HC-067047 greatly improve the pneumonedema, the lung pathologic changes, the up-regulation of proinflammatory cytokines and the neutrophil infiltration in LPS-induced lung injury. In vitro, knockdown of TRPV4 in macrophages reduces the levels of pro-inflammatory cytokines, ROS production, Ca2+ concentration in cytoplasma and the activation of calcineurin/NFATc3 signaling. Importantly, change of extracellular Ca2+ in culture medium prevents LPS-induced NFATc3 nuclear translocation, up-regulation of proinflammatory cytokines and ROS production in macrophages. Inhibition of calcineurin with cyclosporine A, FK506 down-regulates the levels of NFATc3 nuclear translocation and proinflammatory cytokines expression. Our results demonstrate that TRPV4-dependent Ca2+ influx contributes to LPS-induced macrophage activation by calcineurin-NFATc3 pathway.

P3198Long noncoding RNA NEAT1 controls ROS production in macrophages and is suppressed in post-myocardial infarction patients

P3198Long noncoding RNA NEAT1 controls ROS production in macrophages and is suppressed in post-myocardial infarction patients

Myocardin-related transcription factor A (MRTF-A) contributes to acute kidney injury by regulating macrophage ROS production

A host of pathogenic factors induce acute kidney injury (AKI) leading to insufficiencies of renal function. In the present study we evaluated the role of myocardin-related transcription factor A (MRTF-A) in the pathogenesis of AKI. We report that systemic deletion of MRTF-A or inhibition of MRTF-A activity with CCG-1423 significantly attenuated AKI in mice induced by either ischemia-reperfusion or LPS injection. Of note, MRTF-A deficiency or suppression resulted in diminished renal ROS production in AKI models with down-regulation of NAPDH oxdiase 1 (NOX1) and NOX4 expression. In cultured macrophages, MRTF-A promoted NOX1 transcription in response to either hypoxia-reoxygenation or LPS treatment. Interestingly, macrophage-specific MRTF-A deletion ameliorated AKI in mice. Mechanistic analyses revealed that MRTF-A played a role in regulating histone H4K16 acetylation surrounding the NOX gene promoters by interacting with the acetyltransferase MYST1. MYST1 depletion repressed NOX transcription in macrophages. Finally, administration of a MYST1 inhibitor MG149 alleviated AKI in mice. Therefore, we data illustrate a novel epigenetic pathway that controls ROS production in macrophages contributing to AKI. Targeting the MRTF-A-MYST1-NOX axis may yield novel therapeutic strategies to combat AKI.

Immune modulatory properties of 6-gingerol and resveratrol in Atlantic salmon macrophages

The innate immune system provides the first barrier to infection and disease through various processes including activation of inflammation. Inflammation is a biological process whereby immune cells, including macrophages, respond to, and work together to eliminate, damage from injury and disease. Chronic or sustained inflammation, however, can be detrimental to the health and growth of animals, including fish. Immune modulating functional plant compounds, supplemented as feed additives, may be safe and natural approaches to controlling inflammation and disease. This project employed an in vitro cell model to assess the effect of two plant phytoextracts, 6-gingerol and resveratrol, on Atlantic salmon macrophages. To determine if these plant compounds can modify Atlantic salmon macrophage function, primary macrophages were isolated from the head kidney, cultured and pre-treated with the compound for 24 hours. The cells were then challenged with a bacterial pathogen-associated molecular pattern (PAMP), lipopolysaccharide (LPS). To determine if the plant compound pre-treatment modifies macrophage response to LPS stimulation, the mRNA expression of anti-bacterial and cytokine genes (COX2, TNFα, IL-1β, IL-8, Lect-2 and sTLR5) was analyzed by real-time quantitative polymerase chain reaction (qPCR). In addition, the effects of compound pre-treatment on macrophage ROS production and phagocytic responses were analyzed via flow cytometry. We found that both 6-gingerol and resveratrol modified macrophage function, with 6-gingerol having an overall greater effect on expression of the genes analyzed, suggesting that these functional plant compounds may be good candidates as feed additives and should be investigated further.

Altered Glutathione Homeostasis in NLRP3 Inflammasome Activation Mediated by ATP-P2X7 Receptor Signaling

NLRP3 inflammasome is an intracellular protein complex of which activation is responsible for maturation of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-18. Full activation of NLRP3 inflammasome requires two distinct steps; expression of component proteins as well as pro-IL-1beta induced by pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS), followed by complex formation activated by damage-associated molecular patterns (DAMPs) such as ATP. Although redox-dependent regulation of the complex formation has been suggested, it remains unclear how cellular redox status is regulated during ATP stimulation in cells. Here we describe that glutathione (GSH), an antioxidant peptide abundantly present in cells, plays an important role in ATP-dependent inflammasome activation. LC-MSMS analyses clearly demonstrated that cellular levels of GSH were rapidly decline upon ATP stimulation in LPS-primed mouse macrophage J774.1 cells. Similar levels of GSH were detected in the culture supernatants suggesting that ATP stimulated GSH efflux. It should be noted that ATP treatment also induced efflux of polysulfurated derivatives of GSH such as GSSH and GSSSH as well. P2X7 receptor antagonist A-804598 treatment remarkably inhibited both IL-1beta production as well as decrease of GSH. GSH or its oxidized form GSSG added extracellularly suppressed ATP-dependent decline of intracellular GSH, and more importantly, IL-1beta production. Analyses with use of ROS-sensitive probe DCDHF-DA revealed that ATP stimulation augmented ROS production in macrophages, and it was cancelled in the presence of GSSG. Thus, macrophages use ATP-P2X7 receptor signaling to modulate GSH homeostasis via GSH efflux, thereby regulating redox balance for the inflammasome activation.

NADPH Oxidases Are Essential for Macrophage Differentiation

NADPH oxidases (NOXs) are involved in inflammation, angiogenesis, tumor growth, and osteoclast differentiation. However, the role of NOX1 and NOX2 in macrophage differentiation and tumor progression is still elusive. Here we report that NOX1 and NOX2 are critical for the differentiation of monocytes to macrophages, the polarization of M2-type but not M1-type macrophages, and the occurrence of tumor-associated macrophages (TAMs). We found that deletion of both NOX1 and NOX2 led to a dramatic decrease in ROS production in macrophages and resulted in impaired efficiency in monocyte-to-macrophage differentiation and M2-type macrophage polarization. We further showed that NOX1 and NOX2 were critical for the activation of the MAPKs JNK and ERK during macrophage differentiation and that the deficiency of JNK and ERK activation was responsible for the failure of monocyte-to-macrophage differentiation, in turn affecting M2 macrophage polarization. Furthermore, we demonstrated that the decrease in M2 macrophages and TAMs, concomitant with the reduction of cytokine and chemokine secretion, contributed to the delay in wound healing and the inhibition of tumor growth and metastasis in NOX1/2 double knockout mice compared with WT mice. Collectively, these data provide direct evidence that NOX1 and NOX2 deficiency impairs macrophage differentiation and the occurrence of M2-type TAMs during tumor development.

The adhesion GPCR BAI1 mediates macrophage ROS production and microbicidal activity against Gram-negative bacteria.

The detection of microbes and initiation of an innate immune response occur through pattern recognition receptors (PRRs), which are critical for the production of inflammatory cytokines and activation of the cellular microbicidal machinery. In particular, the production of reactive oxygen species (ROS) by the NADPH oxidase complex is a critical component of the macrophage bactericidal machinery. We previously characterized brain-specific angiogenesis inhibitor 1 (BAI1), a member of the adhesion family of G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors (GPCRs), as a PRR that mediates the selective phagocytic uptake of Gram-negative bacteria by macrophages. We showed that BAI1 promoted phagosomal ROS production through activation of the Rho family guanosine triphosphatase (GTPase) Rac1, thereby stimulating NADPH oxidase activity. Primary BAI1-deficient macrophages exhibited attenuated Rac GTPase activity and reduced ROS production in response to several Gram-negative bacteria, resulting in impaired microbicidal activity. Furthermore, in a peritoneal infection model, BAI1-deficient mice exhibited increased susceptibility to death by bacterial challenge because of impaired bacterial clearance. Together, these findings suggest that BAI1 mediates the clearance of Gram-negative bacteria by stimulating both phagocytosis and NADPH oxidase activation, thereby coupling bacterial detection to the cellular microbicidal machinery.

Molecular network-based analysis of guizhi-shaoyao-zhimu decoction, a TCM herbal formula, for treatment of diabetic peripheral neuropathy.

Guizhi-shaoyao-zhimu decoction (GSZ), a traditional Chinese medicine (TCM) herbal formula, has been shown effective in the treatment of diabetic peripheral neuropathy (DPN). In this study, network analysis was performed to decipher the molecular mechanisms of GSZ in the treatment of DPN. The chemical components of the 3 herbs forming GSZ, ie, Ramulus Cinnamomi (Guizhi), Paeonia lactiflora (Shaoyao) and Rhizoma Anemarrhenae (Zhimu), were searched in Chinese medicine dictionaries, and their target proteins were identified in PubChem. DPN genes were searched in PubMed gene databases. Ingenuity Pathway Analysis (IPA) was used to build the GSZ pharmacological network and DPN molecular network. The canonical pathways between the two networks were compared to decipher the molecular mechanisms of GSZ in the treatment of DPN. Sixty-one protein targets for Guizhi, 31 targets for Shaoyao, 47 targets for Zhimu, as well as 23 genes related to DPN were identified and uploaded to IPA. The primary functions of the DPN molecular network were inflammatory response, metabolic disease, cellular assembly and organization. As far as the pharmacological network functions were concerned, Guizhi target proteins were involved in neurological disease, inflammatory disease, cellular growth and proliferation, cell signaling, molecular transport, and nucleic acid metabolism, Shaoyao target proteins were related to neurological disease, inflammatory disease, and Zhimu target proteins focused on cell death and survival, cellular movement, immune cell trafficking, DNA replication, recombination and repair, and cell cycle. In the three-herb combination GSZ, several new network functions were revealed, including the inflammatory response, gene expression, connective tissue development and function, endocrine system disorders, and metabolic disease. The canonical pathway comparison showed that Shaoyao focused on IL-12 signaling and production in macrophages, and Zhimu focused on TNFR2 signaling, death receptor signaling, ILK signaling, IL-17A in gastric cells, IL-6 signaling, IL-8 signaling, the role of JAK1, JAK2, and TYK2 in interferon signaling, IL-9 signaling, HMGB1 signaling, NO production and ROS production in macrophages, whereas GSZ focused aryl hydrocarbon receptor signaling and apoptosis signaling in addition to those pathways induced by Guizhi, Shaoyao and Zhimu. Although each single herb can affect some DPN-related functions and pathways, GSZ exerts more effects on DPN-related functions and pathways. The effects of GSZ on aryl hydrocarbon receptor signaling and apoptosis signaling pathways may be the key components of its total molecular mechanisms.

Macrophage NADPH Oxidase Activation and ROS Production Is Positively Regulated By Shp2 Phosphatase Function

Macrophages are professional phagocytic cells, and express pattern recognition receptors such as C-type lectins and integrins for the detection of invading pathogens. Both Dectin-1 (a C-type lectin) and complement receptor 3 (CR3, a β2-integrin) are expressed on innate immune cells including macrophages, neutrophils, and dendritic cells. Dectin-1 stimulation by b-glucan-containing particles (zymosan) and CR3 stimulation by serum opsonized zymosan (SOZ) activate Erk- and Akt-dependent signaling resulting in phagocytosis and production of an oxidative burst.Shp2, a protein tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt signaling, supports hematopoietic development, and is commonly mutated in juvenile myelomonocytic leukemia (JMML). However, no studies have examined the role of Shp2 in Dectin-1- or CR3-stimulated NADPH oxidase activation or ROS production. As activation of Erk and Akt stimulates NADPH oxidase by phosphorylating p47phox, we hypothesized that Shp2 positively regulates ROS production in response to Dectin-1 or CR3 stimulation.Using murine peritoneal exudate macrophages (PEMs), both zymosan and SOZ exposure induced maximal ROS production 10 minutes post-stimulation, which corresponded to maximal induction of Shp2 phosphorylation (Y580, proposed to promote Shp2 phosphatase activity) and Erk phosphorylation. Using bone marrow derived macrophages (BMMs) from mice bearing a conditionally deleted allele of Ptpn11 (Shp2flox/flox;Mx1Cre+), ROS production was significantly reduced in response to zymosan and SOZ in Shp2flox/flox;Mx1Cre+ BMMs compared to control Shp2flox/flox;Mx1Cre- BMMs. Notably, the phagocytic index of the Shp2flox/flox;Mx1Cre+ and Shp2flox/flox;Mx1Cre- BMMs was similar, and protein components of the NADPH oxidase complex (p40phox, p67phox, and p47phox) were expressed at similar levels.To define the biochemical role of Shp2 in ROS production, we generated yellow fluorescent protein (YFP)-tagged Shp2 constructs bearing mutation of the N-SH2 (R32K) or phosphatase (C463A) domain and retrovirally expressed these constructs in murine BMMs. When subjected to zymosan or SOZ stimulation, mutation of either the N-SH2 or phosphatase domain resulted in reduced ROS production. Using time-lapse confocal videomicroscopy, we found that Shp2-R32K-YFP failed to translocate to the phagosome in SOZ-stimulated BMMs; however, phosphatase dead Shp2-C463A-YFP strongly translocated to the phagosome despite producing lower ROS levels. These findings specifically pointed to Shp2 phosphatase function as crucial in positively regulating NADPH oxidase and ROS production. Accordingly, we reasoned that macrophages expressing JMML-associated gain-of-function (GOF) Shp2 mutants, characterized to have increased phosphatase activity, would produce elevated ROS levels. As anticipated, BMMs retrovirally expressing GOF Shp2-D61Y or GOF Shp2-E76K and PEMs from mice bearing a conditionally induced gain-of-function allele of Ptpn11 (Shp2D61Y/+;Mx1Cre+) similarly produced significantly elevated levels of zymosan- and SOZ-stimulated ROS compared to WT Shp2-expressing BMMs or PEMs, respectively.Given the positive role of Shp2 phosphatase in promoting zymosan- and SOZ-stimulated ROS production, we investigated putative Shp2 substrates in response to zymosan stimulation. SHPS-1 (SH2 domain-containing protein tyrosine phosphatase substrate 1) is a myeloid inhibitory immunoreceptor expressed on macrophages, requires tyrosine phosphorylation to exert its inhibitory effect, and has been shown to be de-phosphorylated by Shp2. Consistent with its potential function in regulation ROS production, SHPS-1 is strongly associated with phagosomes in zymosan-stimulated PEMs. In immunoblot analysis, reduced phospho-SHPS-1 levels kinetically correlated with maximal zymosan-stimulated Shp2 phosphorylation and ROS production, and increased levels of phospho-SHPS-1 were found in BMMs expressing phosphatase dead Shp2-C463A compared to cells expressing WT Shp2. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated NADPH oxidase activation and ROS production in macrophages, and that mechanistically, Shp2 may exert its positive effect by de-phosphorylating and thus negatively regulating the inhibitory function of SHPS-1. DisclosuresNo relevant conflicts of interest to declare.

Ethanol‐induced ROS production is prevented by globular adiponectin via modulation of Nox2 expression in macrophages (844.19)

Adiponectin predominantly secreted from adipose tissue plays a crucial role in lipid metabolism and possesses potent anti‐inflammatory properties. Adipoonectin has been shown to generate protective effects in a number of pathophysiological states. Recently, it has been shown that adiponectin prevents ROS production induced by stimulation with various stimuli. In the present study, we investigated the effect of globular adiponectin (gAcrp) on ethanol‐induced ROS production in macrophages and underlying mechanisms. Herein, we found that gAcrp efficiently suppresses ethanol‐induced ROS production both in RAW 264.7 macrophages and murine peritoneal macrophages. We next investigated the role of NADPH oxidase, comprising a multi‐component enzyme complex, in the suppression of ROS production by gAcrp, since NADPH oxidase plays a critical role in ethanol‐induced ROS production in macrophages. Globular adiponectin prevented ethanol‐induced NAPDH oxidase activation. Particularly, it inhibited ethanol‐induced increase in expression of Nox2 and p22phox. Furthermore, gAcrp restored ethanol‐induced activation of p38MAPK‐NF‐κB pathway, which is critical for Nox2 expression. Taken together, these results suggest that adiponectin prevents ethanol‐induced ROS production via modulation of NADPH oxidase in macrophages.

Ethanol increases matrix metalloproteinase-12 expression via NADPH oxidase-dependent ROS production in macrophages

Matrix metalloproteinase-12 (MMP-12), an enzyme responsible for degradation of extracellular matrix, plays an important role in the progression of various diseases, including inflammation and fibrosis. Although most of those are pathogenic conditions induced by ethanol ingestion, the effect of ethanol on MMP-12 has not been explored. In the present study, we investigated the effect of ethanol on MMP-12 expression and its potential mechanisms in macrophages. Here, we demonstrated that ethanol treatment increased MMP-12 expression in primary murine peritoneal macrophages and RAW 264.7 macrophages at both mRNA and protein levels. Ethanol treatment also significantly increased the activity of nicotinamide adenine dinucleotide (NADPH) oxidase and the expression of NADPH oxidase-2 (Nox2). Pretreatment with an anti-oxidant (N-acetyl cysteine) or a selective inhibitor of NADPH oxidase (diphenyleneiodonium chloride (DPI)) prevented ethanol-induced MMP-12 expression. Furthermore, knockdown of Nox2 by small interfering RNA (siRNA) prevented ethanol-induced ROS production and MMP-12 expression in RAW 264.7 macrophages, indicating a critical role for Nox2 in ethanol-induced intracellular ROS production and MMP-12 expression in macrophages. We also showed that ethanol-induced Nox2 expression was suppressed by transient transfection with dominant negative IκB-α plasmid or pretreatment with Bay 11-7082, a selective inhibitor of NF-κB, in RAW 264.7 macrophages. In addition, ethanol-induced Nox2 expression was also attenuated by treatment with a selective inhibitor of p38 MAPK, suggesting involvement of p38 MAPK/NF-κB pathway in ethanol-induced Nox2 expression. Taken together, these results demonstrate that ethanol treatment elicited increase in MMP-12 expression via increase in ROS production derived from Nox2 in macrophages.

Phospholipase Cγ2 (PLCγ2) Is Key Component in Dectin-2 Signaling Pathway, Mediating Anti-fungal Innate Immune Responses

C-type lectin receptors (CLRs) such as Dectin-2 function as pattern recognition receptors to sense fungal infection. However, the signaling pathways induced by these receptors remain largely unknown. Previous studies suggest that the CLR-induced signaling pathway may utilize similar signaling components as the B cell receptor-induced signaling pathway. Phospholipase Cγ2 (PLCγ2) is a key component in B cell receptor signaling, but its role in other signaling pathways has not been fully characterized. Here, we show that PLCγ2 functions downstream of Dectin-2 in response to the stimulation by the hyphal form of Candida albicans, an opportunistic pathogenic fungus. Using PLCγ2- and PLCγ1-deficient macrophages, we found that the lack of PLCγ2, but not PLCγ1, impairs cytokine production in response to infection with C. albicans. PLCγ2 deficiency results in the defective activation of NF-κB and MAPK and a significantly reduced production of reactive oxygen species following fungal challenge. In addition, PLCγ2-deficient mice are defective in clearing C. albicans infection in vivo. Together, these findings demonstrate that PLCγ2 plays a critical role in CLR-induced signaling pathways, governing antifungal innate immune responses.