Filamentous fungi produce several beneficial secondary metabolites, including bioactive compounds, food additives, and biofuels. Trichoderma, which is a teleomorphic Hypocrea that falls under the taxonomic groups Ascomycota and Dikarya, is an extensively studied fungal genus. In an ongoing study that seeks to discover bioactive natural products, we investigated potential bioactive metabolites from the methanolic extract of cultured Trichoderma gamsii. Using liquid chromatography-mass spectrometry (LC-MS), one major compound was isolated and structurally identified as 6-pentyl-α-pyrone (6PP) based on nuclear magnetic resonance data and LC-MS analysis. To determine its antioxidant and anti-inflammatory activity, as well as the underlying mechanisms, we treated lipopolysaccharide (LPS)-stimulated Raw264.7 mouse macrophages with 6PP. We found that 6PP suppresses LPS-induced increase in the levels of nitric oxide, a mediator of oxidative stress and inflammation, and restores LPS-mediated depletion of total glutathione by stabilizing nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidative factor, and elevating heme oxygenase-1 levels. Furthermore, 6PP inhibited LPS-induced production of proinflammatory cytokines, which are, at least in part, regulated by heme oxygenase-1 (HO-1). 6PP suppressed proinflammatory responses by inhibiting the nuclear localization of nuclear factor kappa B (NF-κB), as well as by dephosphorylating the mitogen-activated protein kinases (MAPKs). These results indicate that 6PP can protect macrophages against oxidative stress and LPS-induced excessive inflammatory responses by activating the Nrf2/HO-1 pathway while inhibiting the proinflammatory, NF-κB, and MAPK pathways.

Ehretia tinifolia (E. tinifolia) L., an evergreen tree with substantial biological activity, including antioxidant and anti-inflammatory effects, has been used in many herbal and traditional medicines. To elucidate its antioxidant and anti-inflammatory activity and the underlying mechanisms, we applied a methanol extract of E. tinifolia (ETME) to lipopolysaccharide (LPS)-stimulated mouse immortalized Kupffer cells. ETME suppressed the LPS-induced increase in nitric oxide, a mediator for oxidative stress and inflammation, and restored LPS-mediated depletion of total glutathione level by stabilizing antioxidative nuclear factor erythroid 2-related factor 2 (Nrf2) and the subsequent increase in heme oxygenase-1 levels. Furthermore, ETME inhibited the LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The inhibitory effects of ETME on pro-inflammatory responses were regulated by ETME-mediated dephosphorylation of mitogen-activated protein kinases (MAPKs: p38, p44/p42, and stress-associated protein kinase/c-Jun N-terminal kinase) and inhibition of nuclear localization of nuclear factor kappa B (NF-κB). These results suggest that ETME is a possible candidate for protecting Kupffer cells from LPS-mediated oxidative stress and excessive inflammatory responses by activating antioxidant Nrf2/HO-1 and inhibiting pro-inflammatory NF-κB and MAPKs, respectively.

The high-affinity IgE receptor FcεRI is the fundamental mast cell (MC) receptor responsible for the involvement of MCs in IgE-associated allergic disorders. Activation of the FcεRI is achieved via crosslinking by multivalent antigen (Ag) recognized by IgE, which results in degranulation and pro-inflammatory cytokine production. In comparison to the T and B cell receptor complexes, for which several co-receptors that orchestrate the initial signaling have been described, information is scarce about FcεRI-associated proteins. Additionally, it is not completely clear how FcεRI signaling synergizes with input from other receptors and how potential regulators affect this synergistic response. We aimed at identifying new regulators of FcεRI and found that the HDL receptor SR-BI (gene name: Scarb1 / SCARB1 ) is expressed in MCs, functionally associates with FcεRI and regulates the local plasma membrane cholesterol content in cholesterol-rich plasma membrane nanodomains as shown by using the cholesterol-sensitive probe GFP-D4. This impacted on the activation of murine MCs upon co-stimulation of the FcεRI with different receptors known to synergize with FcεRI-signaling pathways. Amongst them we investigated the co-activation of the FcεRI with the receptor tyrosine kinase KIT, the IL-33 receptor and GPCRs activated by adenosine or PGE 2 . Scarb1 -deficient bone marrow-derived MCs (BMMCs) showed reduced cytokine secretion in response to these co-stimulation conditions suggesting a role for plasma membrane-associated cholesterol regulating MC-driven inflammation. Mimicking Scarb1 deficiency by membrane cholesterol depletion employing MβCD, we identified PKB and PLCγ1 as cholesterol-sensitive signaling molecules activated downstream of FcεRI in BMMCs. Specifically, when murine MCs were stimulated with SCF and Ag in combination, PLCγ1 activation appeared to be drastically boosted and this could be mitigated by cholesterol depletion. Inhibiting SR-BI in BMMCs phenocopied this effect. Similarly, SR-BI inhibition also attenuated the synergistic response to PGE 2 and anti-IgE in the human ROSA KIT WT mast cell line suggesting that SR-BI is a crucial regulator of synergistic MC activation by regulating the local plasma membrane cholesterol concentration.

Acute myocardial infarction (MI) results in loss of cardiomyocytes and abnormal cardiac remodeling with severe inflammation and fibrosis. However, how cardiac repair can be achieved by timely resolution of inflammation and cardiac fibrosis remains incompletely understood. Our previous findings have shown that dual-specificity phosphatase 6 (DUSP6) is a regeneration repressor from zebrafish to rats. In this study, we found that intravenous administration of the DUSP6 inhibitor (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI) improved heart function and reduced cardiac fibrosis in MI rats. Mechanistic analysis revealed that BCI attenuated macrophage inflammation through NF-κB and p38 signaling, independent of DUSP6 inhibition, leading to the downregulation of various cytokines and chemokines. In addition, BCI suppressed differentiation-related signaling pathways and decreased bone-marrow cell differentiation into macrophages through inhibiting DUSP6. Furthermore, intramyocardial injection of poly (D, L-lactic-co-glycolic acid)-loaded BCI after MI had a notable effect on cardiac repair. In summary, BCI improves heart function and reduces abnormal cardiac remodeling by inhibiting macrophage formation and inflammation post-MI, thus providing a promising pro-drug candidate for the treatment of MI and related heart diseases. This article has an associated First Person interview with the first author of the paper.© 2022. Published by The Company of Biologists Ltd.

Mast cells (MCs) are immune cells of the myeloid lineage distributed in tissues throughout the body. Phenotypically, they are a heterogeneous group characterized by different protease repertoires stored in secretory granules and differential presence of receptors. To adequately address aspects of MC biology either primary MCs isolated from human or mouse tissue or different human MC lines, like HMC-1.1 and -1.2, or rodent MC lines like L138.8A or RBL-2H3 are frequently used. Nevertheless, cellular systems to study MC functions are very limited. We have generated a murine connective tissue-like MC line, termed PMC-306, derived from primary peritoneal MCs (PMCs), which spontaneously transformed. We analyzed PMC-306 cells regarding MC surface receptor expression, effector functions and respective signaling pathways, and found that the cells reacted very similar to primary wildtype (WT) PMCs. In this regard, stimulation with MAS-related G-protein-coupled receptor member B2 (MRGPRB2) ligands induced respective signaling and effector functions. Furthermore, PMC-306 cells revealed significantly accelerated cell cycle progression, which however was still dependent on interleukine 3 (IL-3) and stem cell factor (SCF). Phenotypically, PMC-306 cells adopted an immature connective tissue-like MCs appearance. The observation of cellular transformation was accompanied by the loss of Cdkn2a and Arf expression, which are both described as critical cell cycle regulators. The loss of Cdkn2a and Arf expression could be mimicked in primary bone marrow-derived mast cells (BMMCs) by sustained SCF supplementation strongly arguing for an involvement of KIT activation in the regulation of Cdkn2a/Arf expression. Hence, this new cell line might be a useful tool to study further aspects of PMC function and to address tumorigenic processes associated with MC leukemia.
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