Five out of eight human glutathione peroxidases (GPXs) are selenoproteins, representing proteins that contain selenium as part of the amino acid selenocysteine. The GPXs are important for reducing hydroperoxides in a glutathione-consuming manner and thus regulate cellular redox homeostasis. GPX1, GPX2, and GPX4 represent the three main cytosolic GPXs, but they differ in their expression patterns with GPX1 and GPX4 being expressed ubiquitously, whereas GPX2 is mainly expressed in epithelial cells. GPX1 and GPX2 have been described to reduce soluble hydroperoxides, while GPX4 reduces complex lipid hydroperoxides, thus protecting cells from lipid peroxidation and ferroptosis. But most of these data are derived from cells that are devoid of one of the isoforms and thus, compensation or other cellular effects might affect the conclusions. So far, the use of isolated recombinant human selenoprotein glutathione peroxidases in pure enzyme assays has not been employed to study their substrate specificities side by side. Using recombinant GPX1, GPX2, and GPX4 produced in E. coli we here assessed their GPX activities by a NADPH-consuming glutathione reductase-coupled assay with 17 different peroxides (all at 50 μM) as substrates. GPX4 was clearly the only isoform able to reduce phosphatidylcholine hydroperoxide. In contrast, small soluble hydroperoxides such as H₂O₂, cumene hydroperoxide, and tert-butyl hydroperoxide were reduced by all three isoforms, but with approximately 10-fold higher efficiency for GPX1 in comparison to GPX2 and GPX4. Also, several fatty acid-derived hydroperoxides were reduced by all three isoforms and again GPX1 had the highest activity. Interestingly, the stereoisomerism of the fatty acid-derived hydroperoxides clearly affected the activity of the GPX enzymes. Overall, distinct substrate specificity is obvious for GPX4, but not so when comparing GPX1 and GPX2. Clearly GPX1 was the most potent isoform of the three GPXs in terms of turnover in reduction of soluble and fatty-acid derived hydroperoxides.

Aging is characterized by alterations in the inflammatory microenvironment, which is tightly regulated by a complex network of inflammatory mediators. Excessive calorie consumption contributes to age- and lifestyle-associated diseases like obesity, type 2 diabetes, cardiovascular disorders, and cancer, while limited nutrient availability may lead to systemic health-promoting adaptations. Geroprotective effects of short-term caloric restriction (CR) can beneficially regulate innate immune receptors and interferon signaling in the liver of aged mice, but how CR impacts the hepatic release of immunomodulatory mediators like cytokines and lipid mediators (LM) is elusive. Here, we investigated the impact of aging on the inflammatory microenvironment in the liver and its linkage to calorie consumption. The livers of female young and aged C57BL/6JRj mice, as well as of aged mice after caloric restriction (CR) up to 28 days, with and without subsequent re-feeding (2 days), were evaluated. Surprisingly, despite differences in the hepatic proteome of young and old mice, aging did not promote a pro-inflammatory environment in the liver, but it reduced lipoxygenase-mediated formation of LM from polyunsaturated fatty acids without affecting the expression of the involved lipoxygenases and related oxygenases. Moreover, CR failed to ameliorate the secretion of pro-inflammatory cytokines but shifted the LM production to the formation of monohydroxylated LM with inflammation-resolving features. Unexpectedly, re-feeding after CR even further decreased the inflammatory response as LM species were markedly downregulated. Our findings raise the question of how short-term CR is indeed beneficial as a nutritional intervention for healthy elderly subjects and further stress the necessity to address tissue-specific inflammatory states.

Specialized pro-resolving mediators (SPM) are di- or trihydroxylated polyunsaturated fatty acids with potent inflammation-resolving features. SPM typically occur at lower concentrations than classical pro-inflammatory eicosanoids, making their detection highly challenging. Thus, reporting of biological SPM levels has been discrepant and not always transparent, sparking uncertainty concerning the formation capacity for di- and especially for trihydroxylated SPM in vitro and in vivo. Here, we create common ground by providing a systematic and comprehensive assessment of SPM formation across a broad range of commonly employed in vitro, ex vivo, and in vivo matrices. For this purpose, a quantitative UHPLC-MS/MS method targeting 72 oxylipins (including 19 SPM) was validated in accordance with recommendations by the International Lipidomics Society. By including sample preparation (solid phase extraction) in all validation processes, a conservative lower limit of quantification (10-100 pg/mL matrix) was found to meet clearly defined criteria for signal/noise, accuracy, precision, selectivity, specificity, matrix effects, recovery, and carry-over for 19 SPM. Our results offer analytically defensible reference data to demonstrate (i) the absence of relevant SPM levels in stimulated whole blood and unstimulated cell models; (ii) only trace SPM formation in stimulated peripheral blood mononuclear cells, M1-macrophages, neutrophils, and platelet incubations; but (iii) significant SPM formation in stimulated M2a-macrophages and neutrophil/platelet co-incubations. In healthy C57BL/6JRj mice, SPM formation was low yet organ-specific, with relevant amounts of dihydroxylated SPM detected in spleen. Taken together, we demonstrate SPM formation to be robust, matrix- and stimulus-dependent, but confined to specific di- and trihydroxylated SPM.

Aging is a systemic immunomodulatory condition that makes organisms more susceptible to infection-driven periodontitis and associated periodontal tissue loss. Therefore, modulation of the bacterial-induced inflammatory host response could potentially target the pathophysiological systemic and local crosstalk and resulting tissue homeostasis in aged organisms. However, underlying pathways connecting nutritional modulation with periodontal disease pathology and whether an aged organism benefits from specific immunomodulatory nutritional components are not known. Based on its potent immunomodulatory function, we determined whether nutritional interventions with dietary monounsaturated fatty acid (FA) oleic acid (OA [C18:1]), a main component of Mediterranean-style diets, as compared with a Western-style diet component saturated FA palmitic acid (PA [C16:0]) could modify the response to periodontal infection in a murine periodontal Porphyromonas gingivalis –inoculation model comparing old (final age of up to 2 y) and young (21 wk) mice. Local fibroblastic response and circulating regulatory immune-competent lipid mediators (LMs) were determined to provide insights into underlying pathways and how diet and age affect inflammation and resolution of the periodontal infection. The dietary intake of PA enhances periodontal bone destruction in response to P. gingivalis infection in old mice, whereas an OA-enriched diet (ED) has a protective effect. Specifically, PA-ED enhances the inflammatory profile of the periodontal microenvironment and primes gingival fibroblasts, prominent regulators within local tissue homeostasis, toward an inflammatory phenotype. Moreover, systems-wide serological lipidomic analyses of LMs and their respective stimulation of osteoclast differentiation revealed 10-hydroxydocosahexaenoic acid, associated with OA intake, as a protective candidate for age- and nutrition-dependent regulation of osteoclast-driven bone loss. Given the rising prevalence of periodontitis in the aging population, incorporating OA-rich foods could offer promising strategies for addressing the sequelae of aging and promoting periodontal health.

Oxylipins, signalling molecules derived from polyunsaturated fatty acids, act as key mediators controlling inflammatory processes. Ageing fuels the disruption of this network, promoting inflammageing. Social isolation, a common feature of ageing, may contribute to the emergence of pro-inflammatory responses, further aggravating conditions like cognitive decline and frailty. Here, we studied how repeated social isolation impacts inflammation-related oxylipin profiles in seven different organs and serum of aged mice. Additionally, we explored physical exercise as a tool to ameliorate age- and isolation-associated inflammation. Our results show that social isolation induces significant increases in interleukin-1β levels and stimulates a dramatic production of lipoxygenase (LOX)-derived oxylipins in an organ-dependent manner, particularly pronounced in liver, lung, and spleen. Physical exercise failed to mitigate the pro-inflammatory effects induced by social isolation. These effects did not occur in the circulatory system as serum oxylipin levels remained relatively unchanged by isolation. The unexpected and striking elevation of oxylipins across the organs highlights the detrimental effect of social isolation and proposes key roles of oxylipins in stress-related inflammageing.