Short-Term Caloric Restriction and Subsequent Re-Feeding Compromise Liver Health and Associated Lipid Mediator Signaling in Aged Mice.

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.

Metabololipidomic and proteomic profiling reveals aberrant macrophage activation and interrelated immunomodulatory mediator release during aging.

Macrophages adapt distinct pro-inflammatory (M1-like) and pro-resolving (M2-like) phenotypes with specific tasks in the immune response and tissue homeostasis. Altered macrophage responses with age are causative for unresolved inflammation, so-called inflammaging, and lead to higher infection susceptibility with unfavorable progression. Here, we reveal molecular determinants of age-related changes in phenotypic functions of murine peritoneal macrophages (PM) by employing comprehensive mass spectrometry-based proteomics (4746 protein groups) and metabololipidomics (>40 lipid mediators). Divergent expression of various macrophage-specific marker proteins and signaling pathways indicates aberrant PM phenotypes in old mice which detrimentally impact their capabilities to release immunomodulatory chemokines and cytokines. We show that aging strikingly compromises the polarization process of macrophages to adapt either pro-inflammatory or pro-resolving phenotypes, thereby yielding aberrant and afunctional macrophage subtypes that cannot be readily assigned to either a typical M1 or M2 phenotype. In particular, the phenotypic adaptation of the bacteria-challenged metabololipidome in macrophages related to inflammation is severely limited by age, which persists across ex vivo polarization towards M1 and M2a macrophages. Our results establish distinct age-associated PM phenotypes outside of the simplified M1 and M2 dichotomy and challenge the dogma of increased pro-inflammatory macrophage pre-activation due to aging by revealing maladaptive functions throughout all phases of inflammation, including resolution.

Inflammaging is driven by upregulation of innate immune receptors and systemic interferon signaling and is ameliorated by dietary restriction.

Aging is characterized by a chronic low-grade inflammation known as inflammaging in multiple tissues, representing a risk factor for age-related diseases. Dietary restriction (DR) is the best-known non-invasive method to ameliorate aging in many organisms. However, the molecular mechanism and the signaling pathways that drive inflammaging across different tissues and how they are modulated by DR are not yet understood. Here we identify a multi-tissue gene network regulating inflammaging. This network is characterized by chromatin opening and upregulation in the transcription of innate immune system receptors and by activation of interferon signaling through interferon regulatory factors, inflammatory cytokines, and Stat1-mediated transcription. DR ameliorates aging-induced alterations of chromatin accessibility and RNA transcription of the inflammaging gene network while failing to rescue those alterations on the rest of the genome. Our results present a comprehensive understanding of the molecular network regulating inflammation in aging and DR and provide anti-inflammaging therapeutic targets.

Metabolic determination of cell fate through selective inheritance of mitochondria.

Metabolic characteristics of adult stem cells are distinct from their differentiated progeny, and cellular metabolism is emerging as a potential driver of cell fate conversions1-4. How these metabolic features are established remains unclear. Here we identified inherited metabolism imposed by functionally distinct mitochondrial age-classes as a fate determinant in asymmetric division of epithelial stem-like cells. While chronologically old mitochondria support oxidative respiration, the electron transport chain of new organelles is proteomically immature and they respire less. After cell division, selectively segregated mitochondrial age-classes elicit a metabolic bias in progeny cells, with oxidative energy metabolism promoting differentiation in cells that inherit old mitochondria. Cells that inherit newly synthesized mitochondria with low levels of Rieske iron-sulfur polypeptide 1 have a higher pentose phosphate pathway activity, which promotes de novo purine biosynthesis and redox balance, and is required to maintain stemness during early fate determination after division. Our results demonstrate that fate decisions are susceptible to intrinsic metabolic bias imposed by selectively inherited mitochondria.

Identifying Cell-Type-Specific Metabolic Signatures Using Transcriptome and Proteome Analyses.

Studies in various tissues have revealed a central role of metabolic pathways in regulating adult stem cell function in tissue regeneration and tumor initiation. The unique metabolic dependences or preferences of adult stem cells, therefore, are emerging as a new category of therapeutic target. Recently, advanced methods including high-resolution metabolomics, proteomics, and transcriptomics have been developed to address the growing interest in stem cell metabolism. A practical framework integrating the omics analyses is needed to systematically perform metabolic characterization in a cell-type-specific manner. Here, we leverage recent advances in transcriptomics and proteomics research to identify cell-type-specific metabolic features by reconstructing cell identity using genes and the encoded enzymes involved in major metabolic pathways. We provide protocols for cell isolation, transcriptome and proteome analyses, and metabolite profiling and measurement. The workflow for mapping cell-type-specific metabolic signatures presented here, although initially developed for intestinal crypt cells, can be easily implemented for cell populations in other tissues, and is highly compatible with most public datasets. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Intestinal crypt isolation and cell population purification Basic Protocol 2: Transcriptome analyses for cell-type-specific metabolic gene expression Basic Protocol 3: Proteome analyses for cell-type-specific metabolic enzyme levels Basic Protocol 4: Metabolite profiling and measurement.

Aging drives organ-specific alterations of the inflammatory microenvironment guided by immunomodulatory mediators in mice.

Aging is accompanied by chronic, low-grade systemic inflammation, termed inflammaging, a main driver of age-associated diseases. Such sterile inflammation is typically characterized by elevated levels of pro-inflammatory mediators, such as cytokines, chemokines and reactive oxygen species causing organ damage. Lipid mediators play important roles in the fine-tuning of both the promotion and the resolution of inflammation. Yet, it remains unclear how lipid mediators fit within the concept of inflammaging and how their biosynthesis and function is affected by aging. Here, we provide comprehensive signature profiles of inflammatory markers in organs afflicted with inflammation of young and old C57BL/6 mice. We reveal an organ-specific footprint of inflammation-related cytokines, chemokines and lipid mediators, which are distinctively affected by aging. While some organs are characterized by a pronounced pro-inflammatory microenvironment and impaired resolution during aging, others display elevated levels of pro-resolving mediators or an overall decrease in inflammatory signaling. Our results demonstrate that it proves difficult to establish a unifying concept for alterations of immunomodulatory mediators as consequence of aging and that organ specificity needs to be considered. Moreover, our data imply that inclusion of lipid mediators into the concept of inflammaging provides a comprehensive tool to characterize the inflammatory microenvironment during aging on a broader and yet, more detailed scope.

Stem cell aging: The upcoming era of proteins and metabolites.

So far, the investigation of stem cell aging has been primarily carried out at the genomic level to follow transcriptome changes, clonal dominance, epigenetic changes, and, more recently, population heterogeneity by single cell sequencing. Here, we review recent findings in the field of stem cell aging that include failure of proteostasis, the impact of age-related metabolic changes on stem cell differentiation and heterogeneity of stem cell niches. These emerging concepts highlight the need of a paradigm shift to move forward our understanding of stem cell aging to the next level, in particular, the need of investigating cell intrinsic changes in stem cells and their niche by looking in a quantitative manner to proteins and metabolites.

Region-Specific Proteome Changes of the Intestinal Epithelium during Aging and Dietary Restriction.

The small intestine is responsible for nutrient absorption and one of the most important interfaces between the environment and the body. During aging, changes of the epithelium lead to food malabsorption and reduced barrier function, thus increasing disease risk. The drivers of these alterations remain poorly understood. Here, we compare the proteomes of intestinal crypts from mice across different anatomical regions and ages. We find that aging alters epithelial immunity, metabolism, and cell proliferation and is accompanied by region-dependent skewing in the cellular composition of the epithelium. Of note, short-term dietary restriction followed by refeeding partially restores the epithelium by promoting stem cell differentiation toward the secretory lineage. We identify Hmgcs2 (3-hydroxy-3-methylglutaryl-coenzyme A [CoA] synthetase 2), the rate-limiting enzyme for ketogenesis, as a modulator of stem cell differentiation that responds to dietary changes, and we provide an atlas of region- and age-dependent proteome changes of the small intestine.

Dietary restriction improves repopulation but impairs lymphoid differentiation capacity of hematopoietic stem cells in early aging.

Dietary restriction (DR) improves health, delays tissue aging, and elongates survival in flies and worms. However, studies on laboratory mice and nonhuman primates revealed ambiguous effects of DR on lifespan despite improvements in health parameters. In this study, we analyzed consequences of adult-onset DR (24 h to 1 yr) on hematopoietic stem cell (HSC) function. DR ameliorated HSC aging phenotypes, such as the increase in number of HSCs and the skewing toward myeloid-biased HSCs during aging. Furthermore, DR increased HSC quiescence and improved the maintenance of the repopulation capacity of HSCs during aging. In contrast to these beneficial effects, DR strongly impaired HSC differentiation into lymphoid lineages and particularly inhibited the proliferation of lymphoid progenitors, resulting in decreased production of peripheral B lymphocytes and impaired immune function. The study shows that DR-dependent suppression of growth factors and interleukins mediates these divergent effects caused by DR. Supplementation of insulin-like growth factor 1 partially reverted the DR-induced quiescence of HSCs, whereas IL-6/IL-7 substitutions rescued the impairment of B lymphopoiesis exposed to DR. Together, these findings delineate positive and negative effects of long-term DR on HSC functionality involving distinct stress and growth signaling pathways.