Epigallocatechin Gallate Regulates the Myeloid-Specific Transcription Factor PU1 in Macrophages

Our previous research demonstrated that PU.1 regulates expression of the genes involved in inflammation in macrophages. Selective knockdown of PU.1 in macrophages ameliorated LPS-induced acute lung injury (ALI) in bone marrow chimera mice. Inhibitors that block the transcriptional activity of PU.1 in macrophages have the potential to mitigate the pathophysiology of LPS-induced ALI. However, complete inactivation of PU.1 gene disrupts normal myelopoiesis. Although the green tea polyphenol Epigallocatechin gallate (EGCG) has been shown to regulate inflammatory genes in various cell types, it is not known if EGCG alters the transcriptional activity of PU.1 protein. Using Schrodinger Glide docking, we have identified that EGCG binds with PU.1 protein, altering its DNA-binding and self-dimerization activity. In silico analysis shows that EGCG forms Hydrogen bonds with Glutamic Acid 209, Leucine 250 in DNA binding and Lysine 196, Tryptophan 193, and Leucine 182 in the self-dimerization domain of the PU.1 protein. Experimental validation using mouse bone marrow-derived macrophages (BMDM) confirmed that EGCG inhibits both DNA binding by PU.1 and self-dimerization. Importantly, EGCG had no impact on expression of the total PU.1 protein levels but significantly reduced expression of various inflammatory genes and generation of ROS. In summary, we report that EGCG acts as an inhibitor of the PU.1 transcription factor in macrophages

The Calcineurin-NFATc Pathway Modulates the Lipid Mediators in BAL Fluid Extracellular Vesicles, Thereby Regulating Microvascular Endothelial Cell Barrier Function

Extracellular vesicles mediate intercellular communication by transporting biologically active macromolecules. Our prior studies have demonstrated that the nuclear factor of activated T cell cytoplasmic member 3 (NFATc3) is activated in mouse pulmonary macrophages in response to lipopolysaccharide (LPS). Inhibition of NFATc3 activation by a novel cell-permeable calcineurin peptide inhibitor CNI103 mitigated the development of acute lung injury (ALI) in LPS-treated mice. Although pro-inflammatory lipid mediators are known contributors to lung inflammation and injury, it remains unclear whether the calcineurin-NFATc pathway regulates extracellular vesicle (EV) lipid content and if this content contributes to ALI pathogenesis. In this study, EVs from mouse bronchoalveolar lavage fluid (BALF) were analyzed for their lipid mediators by liquid chromatography in conjunction with mass spectrometry (LC-MS/MS). Our data demonstrate that EVs from LPS-treated mice contained significantly higher levels of arachidonic acid (AA) metabolites, which were found in low levels by prior treatment with CNI103. The catalytic activity of lung tissue cytoplasmic phospholipase A2 (cPLA2) increased during ALI, correlating with an increased amount of arachidonic acid (AA) in the EVs. Furthermore, ALI is associated with increased expression of cPLA2, cyclooxygenase 2 (COX2), and lipoxygenases (5-LOX, 12-LOX, and 15-LOX) in lung tissue, and pretreatment with CNI103 inhibited the catalytic activity of cPLA2 and the expression of cPLA2, COX, and LOX transcripts. Furthermore, co-culture of mouse pulmonary microvascular endothelial cell (PMVEC) monolayer and NFAT-luciferase reporter macrophages with BALF EVs from LPS-treated mice increased the pulmonary microvascular endothelial cell (PMVEC) monolayer barrier permeability and luciferase activity in macrophages. However, EVs from CNI103-treated mice had no negative impact on PMVEC monolayer barrier integrity. In summary, BALF EVs from LPS-treated mice carry biologically active NFATc-dependent, AA-derived lipids that play a role in regulating PMVEC monolayer barrier function

Targeting ETosis by miR-155 inhibition mitigates mixed granulocytic asthmatic lung inflammation

Asthma is phenotypically heterogeneous with several distinctive pathological mechanistic pathways. Previous studies indicate that neutrophilic asthma has a poor response to standard asthma treatments comprising inhaled corticosteroids. Therefore, it is important to identify critical factors that contribute to increased numbers of neutrophils in asthma patients whose symptoms are poorly controlled by conventional therapy. Leukocytes release chromatin fibers, referred to as extracellular traps (ETs) consisting of double-stranded (ds) DNA, histones, and granule contents. Excessive components of ETs contribute to the pathophysiology of asthma; however, it is unclear how ETs drive asthma phenotypes and whether they could be a potential therapeutic target. We employed a mouse model of severe asthma that recapitulates the intricate immune responses of neutrophilic and eosinophilic airway inflammation identified in patients with severe asthma. We used both a pharmacologic approach using miR-155 inhibitor-laden exosomes and genetic approaches using miR-155 knockout mice. Our data show that ETs are present in the bronchoalveolar lavage fluid of patients with mild asthma subjected to experimental subsegmental bronchoprovocation to an allergen and a severe asthma mouse model, which resembles the complex immune responses identified in severe human asthma. Furthermore, we show that miR-155 contributes to the extracellular release of dsDNA, which exacerbates allergic lung inflammation, and the inhibition of miR-155 results in therapeutic benefit in severe asthma mice. Our findings show that targeting dsDNA release represents an attractive therapeutic target for mitigating neutrophilic asthma phenotype, which is clinically refractory to standard care

EGCG downregulates LPS-inducible PU.1 dependent macrophage gene expression.

A-G) BMDM were pretreated with 5 μM of EGCG for 1 h and stimulated with LPS or PBS for another 4 hours. Expression of IL6, TNFα, TLR4, KC, iNOS, CCR2 and COX2 mRNA levels was determined using gene specific primers and SYBR green reaction mix, as described in methods. H-J) BMDM were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 16 hours and extracellular release of IL6, TNFα, and KC were determined by ELISA. Data are shown as mean ± SEM. N = 5 for each group, A-G ***p H-J ***p < 0.001 DMSO/EGCG/DB2313 vs DMSO+LPS; # p < 0.01 EGCG+LPS or DB2313+LPS vs DMSO+LPS.</p

EGCG alters PU.1 dimerization and DNA binding activity.

A) Total cell lysates from BMDM were incubated in presence of 25 μM EGCG and 1x β-Mercapto ethanol separately and immunoblotted with PU.1 antibody to determine the monomeric or dimeric state. Relative expression levels of COX2, iNOS and β-actin were determined in BMDM pretreated with EGCG, later stimulated with LPS, and B) relative densitometry. C) BMDM nuclear protein extracts were incubated with biotin labeled TLR4 promoter oligo in presence of increasing concentrations of EGCG. The reaction mix was electrophoresed on native polyacrylamide gel in 1X TBE, transferred to Biodyne A membrane and detected by Streptavidn-HRP chemiluminescence detection kit. D) BMDM were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 2 hours. Cells were cross-linked with 1% formaldehyde and PU.1 bound TLR4 promoter was immunoprecipitated, detected by qPCR after sequential washes, de-cross linking and purification steps. PU.1 recruitment on to TLR4 promoter was expressed as % input of chromatin. Graph shows means plus SD for triplicate samples and is representative of 2 independent experiments. Data are shown as mean ± SEM. N = 5 for each group, D **** p < 0.0001 Normal IgG immunoprecipitated chromatin vs Anti-PU.1 IgG immunoprecipitated chromatin.</p

Labeling EGCG with TRITC and determination of EGCG binding affinity.

A) TRITC was conjugated to EGCG as described in methods. EGCG-TRITC was eluted as a single peak by HPLC and the molecular mass of 940 is equivalent to the cumulative mass of EGCG and TRITC together. B) Binding affinity of EGCG-TRITC to PU.1 protein determined by fluorescence anisotropy and calculated dissociation constant of 2.8 μM.</p

EGCG downregulates LPS-inducible PU.1 dependent genes in THP-1 cells.

THP-1 Cells were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 4 hours. Expression of A) IL6 and B) IL8 mRNA levels was determined using gene specific primers and SYBR green reaction mix. THP-1 Cells were pretreated with 5 μM of EGCG or DB2313 for 1 h and stimulated with LPS or PBS for another 16 hours and extracellular release of C) IL8, and D) ROS generation were determined by ELISA and DCFH-DA dye. A-D Data are shown as mean ± SEM. N = 5 for each group, A-C *** p D ** p < 0.01 DMSO/EGCG vs DMSO+LPS; * p < 0.05 EGCG+LPS vs DMSO+LPS.</p

S1 Fig -

Our previous research demonstrated that PU.1 regulates expression of the genes involved in inflammation in macrophages. Selective knockdown of PU.1 in macrophages ameliorated LPS-induced acute lung injury (ALI) in bone marrow chimera mice. Inhibitors that block the transcriptional activity of PU.1 in macrophages have the potential to mitigate the pathophysiology of LPS-induced ALI. However, complete inactivation of PU.1 gene disrupts normal myelopoiesis. Although the green tea polyphenol Epigallocatechin gallate (EGCG) has been shown to regulate inflammatory genes in various cell types, it is not known if EGCG alters the transcriptional activity of PU.1 protein. Using Schrodinger Glide docking, we have identified that EGCG binds with PU.1 protein, altering its DNA-binding and self-dimerization activity. In silico analysis shows that EGCG forms Hydrogen bonds with Glutamic Acid 209, Leucine 250 in DNA binding and Lysine 196, Tryptophan 193, and Leucine 182 in the self-dimerization domain of the PU.1 protein. Experimental validation using mouse bone marrow-derived macrophages (BMDM) confirmed that EGCG inhibits both DNA binding by PU.1 and self-dimerization. Importantly, EGCG had no impact on expression of the total PU.1 protein levels but significantly reduced expression of various inflammatory genes and generation of ROS. In summary, we report that EGCG acts as an inhibitor of the PU.1 transcription factor in macrophages.</div

Molecular docking shows specific interaction of EGCG and mouse PU.1 protein.

A) EGCG was docked on to mouse PU.1 protein 3D model using Schrodinger GLIDE® and protein-ligand structure binding affinity is determined. EGCG establishes hydrogen bonds with Glutamic acid 209, Leucine 250 in the DNA binding domain, and B) Leucine 182, tryptophan 193, lysine 196 in the dimerization domain of PU.1 protein. C) Structural organization of mouse PU.1 protein D) Intercalation of DB2313 with double stranded DNA minor groove.</p