Glucocorticoids regulate lipid mediator networks by reciprocal modulation of 15-lipoxygenase isoforms affecting inflammation resolution.

Glucocorticoids (GC) are potent anti-inflammatory agents, broadly used to treat acute and chronic inflammatory diseases, e.g., critically ill COVID-19 patients or patients with chronic inflammatory bowel diseases. GC not only limit inflammation but also promote its resolution although the underlying mechanisms are obscure. Here, we reveal reciprocal regulation of 15-lipoxygenase (LOX) isoform expression in human monocyte/macrophage lineages by GC with respective consequences for the biosynthesis of specialized proresolving mediators (SPM) and their 15-LOX-derived monohydroxylated precursors (mono-15-OH). Dexamethasone robustly up-regulated pre-mRNA, mRNA, and protein levels of ALOX15B/15-LOX-2 in blood monocyte-derived macrophage (MDM) phenotypes, causing elevated SPM and mono-15-OH production in inflammatory cell types. In sharp contrast, dexamethasone blocked ALOX15/15-LOX-1 expression and impaired SPM formation in proresolving M2-MDM. These dexamethasone actions were mimicked by prednisolone and hydrocortisone but not by progesterone, and they were counteracted by the GC receptor (GR) antagonist RU486. Chromatin immunoprecipitation (ChIP) assays revealed robust GR recruitment to a putative enhancer region within intron 3 of the ALOX15B gene but not to the transcription start site. Knockdown of 15-LOX-2 in M1-MDM abolished GC-induced SPM formation and mono-15-OH production. Finally, ALOX15B/15-LOX-2 upregulation was evident in human monocytes from patients with GC-treated COVID-19 or patients with IBD. Our findings may explain the proresolving GC actions and offer opportunities for optimizing GC pharmacotherapy and proresolving mediator production.

Hyphal-associated protein expression is crucial for Candida albicans-induced eicosanoid biosynthesis in immune cells.

Candida albicans causes opportunistic infections ranging from mucosal mycoses to life-threatening systemic infections in immunocompromised patients. During C. albicans infection, leukotrienes and prostaglandins are formed from arachidonic acid by 5-lipoxygenase (5-LOX) and cyclooxygenases, respectively to amplify inflammatory conditions, but also to initiate macrophage infiltration to achieve tissue homeostasis. Since less is known about the cellular mechanisms triggering such lipid mediator biosynthesis, we investigated the eicosanoid formation in monocyte-derived M1 and M2 macrophages, neutrophils and HEK293 cells transfected with 5-LOX and 5-LOX-activating protein (FLAP) in response to C. albicans yeast or hyphae. Leukotriene biosynthesis was exclusively induced by hyphae in neutrophils and macrophages, whereas prostaglandin E2 was also formed in response to yeast cells by M1 macrophages. Eicosanoid biosynthesis was significantly higher in M1 compared to M2 macrophages. In HEK_5-LOX/FLAP cells only hyphae activated the essential 5-LOX translocation to the nuclear membrane. Using yeast-locked C. albicans mutants, we demonstrated that hyphal-associated protein expression is critical in eicosanoid formation. For neutrophils and HEK_5-LOX/FLAP cells, hyphal wall protein 1 was identified as the essential surface protein that stimulates leukotriene biosynthesis. In summary, our data suggest that hyphal-associated proteins of C. albicans are central triggers of eicosanoid biosynthesis in human phagocytes.

Inhibition of Nox4-dependent ROS signaling attenuates prostate fibroblast activation and abrogates stromal-mediated protumorigenic interactions.

Carcinoma-associated fibroblasts (CAFs) play a key onco-supportive role during prostate cancer (PCa) development and progression. We previously reported that the reactive oxygen species (ROS)-producing enzyme NADPH oxidase 4 (Nox4) is essential for TGFß1-mediated activation of primary prostate human fibroblasts to a CAF-like phenotype. This study aimed to further investigate the functional relevance of prostatic Nox4 and determine whether pharmacological inhibition of stromal Nox4 abrogates paracrine-mediated PCa-relevant processes. RNA in situ hybridization revealed significantly elevated Nox4 mRNA levels predominantly in the peri-tumoral stroma of clinical PCa with intense stromal Nox4 staining adjacent to tumor foci expressing abundant TGFß protein levels. At pharmacologically relevant concentrations, the Nox1/Nox4 inhibitor GKT137831 attenuated ROS production, CAF-associated marker expression and migration of TGFß1-activated but not nonactivated primary human prostate fibroblasts. Similar effects were obtained upon shRNA-mediated silencing of Nox4 but not Nox1 indicating that GKT137831 primarily abrogates TGFß1-driven fibroblast activation via Nox4 inhibition. Moreover, inhibiting stromal Nox4 abrogated the enhanced proliferation and migration of PCa cell lines induced by TGFß1-activated prostate fibroblast conditioned media. These effects were not restricted to recombinant TGFß1 as conditioned media from PCa cell lines endogenously secreting high TGFß1 levels induced fibroblast activation in a stromal Nox4- and TGFß receptor-dependent manner. Importantly, GKT137831 also attenuated PCa cell-driven fibroblast activation. Collectively, these findings suggest the TGFß-Nox4 signaling axis is a key interface to dysregulated reciprocal stromal-epithelial interactions in PCa pathophysiology and provide a strong rationale for further investigating the applicability of Nox4 inhibition as a stromal-targeted approach to complement current PCa treatment modalities.

The α-tocopherol-derived long-chain metabolite α-13'-COOH mediates endotoxin tolerance and modulates the inflammatory response via MAPK and NFκB pathways.

SCOPE: The long-chain metabolites of (LCM) vitamin E are proposed as the active regulatory metabolites of vitamin E providing, with their anti-inflammatory properties, an explanatory approach for the inconsistent effects of vitamin E on inflammatory-driven diseases. We examined the modulation of cytokine expression and release from macrophages, a fundamental process in many diseases, to gain insights into the anti-inflammatory mechanisms of the α-tocopherol-derived LCM α-13'-COOH. METHODS AND RESULTS: Suppressed gene expression of C-C motif chemokine ligand 2 (Ccl2), tumor necrosis factor (Tnf), and interleukin (Il) 6 in response to lipopolysaccharides by 24 h pre-treatment with α-13'-COOH in RAW264.7 macrophages was revealed using quantitative reverse transcription PCR. Further, reduced secretion of IL1ß and CCL2 was found in this setup using flow cytometry. In contrast, 1 h pre-treatment suppressed only CCL2. Consequent gene expression analysis within 24 h of α-13'-COOH treatment revealed the induction of mitogen-activated protein kinases (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) negative feedback regulators including the 'master regulators' dual-specificity phosphatase 1 (Dusp1/Mkp1) and tumor necrosis factor induced protein 3 (Tnfaip3/A20). Approaches with immunoblots and chemical antagonists suggest a feedback induction via activation of extracellular-signal regulated kinase (ERK), p38 MAPK and NFκB pathways. CONCLUSIONS: CCL2 is suppressed in murine macrophages by α-13'-COOH and the indirect suppression of MAPK and NFκB pathways is likely a relevant process contributing to anti-inflammatory actions of α-13'-COOH. These results improve the understanding of the effects of α-13'-COOH and provide a basis for new research strategies in the context of inflammatory diseases.

Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation.

Endogenous long-chain metabolites of vitamin E (LCMs) mediate immune functions by targeting 5-lipoxygenase (5-LOX) and increasing the systemic concentrations of resolvin E3, a specialized proresolving lipid mediator. SAR studies on semisynthesized analogues highlight α-amplexichromanol (27a), which allosterically inhibits 5-LOX, being considerably more potent than endogenous LCMs in human primary immune cells and blood. Other enzymes within lipid mediator biosynthesis were not substantially inhibited, except for microsomal prostaglandin E2 synthase-1. Compound 27a is metabolized by sulfation and ß-oxidation in human liver-on-chips and exhibits superior metabolic stability in mice over LCMs. Pharmacokinetic studies show distribution of 27a from plasma to the inflamed peritoneal cavity and lung. In parallel, 5-LOX-derived leukotriene levels decrease, and the inflammatory reaction is suppressed in reconstructed human epidermis, murine peritonitis, and experimental asthma in mice. Our study highlights 27a as an orally active, LCM-inspired drug candidate that limits inflammation with superior potency and metabolic stability to the endogenous lead.