Project description:Inflammation is the natural defensive response of the immune system to an injury or infection and is regulated by small molecule mediators triggering different phases of the inflammatory process. In particular, lipid mediators (LM) and cytokines exhibit crucial regulatory functions in the progression and resolution of inflammation. Macrophages play a central role in this process and can adopt distinct phenotypes with specialized functions depending on their microenvironment: inflammatory M1 macrophages drive inflammation by the release of pro-inflammatory cytokines and LMs, like prostaglandins (PG) and leukotrienes (LT), while resolving M2 macrophages promote inflammation resolution and tissue regeneration by production of anti-inflammatory cytokines and specialized pro resolving mediators (SPM). Aging is associated with chronic and unresolved, low-grade inflammation (“inflammaging”) and aging-related dysfunction of macrophages in the resolution of inflammation and tissue maintenance has been reported. Yet, the underlying molecular mechanisms and functional consequences of latter processes remain poorly understood. Here, we show that polarization of peritoneal macrophages (PM) from geriatric mice towards an M2-like phenotype is impaired versus adult mice, resulting in aberrant LM formation and cytokine release. In PMs isolated from adult mice (PM-A) we observed a shift in LM formation from PGs and LTs to SPMs already after 4 h of polarization towards M2 with interleukin (IL) 4. In contrast, PMs from geriatric mice (PM-G) produced mainly LTs and PGs upon polarization. This pattern persists over the course of 48 h of polarization. Proteomic profiling revealed that polarization of PM A towards M2 yields a more distinct phenotype, clearly separated from M1, when compared to PM-G. We observed similar aging-related changes in the lipidome and cytokine profile of spleen, lung and liver tissue from mice. Hence, we hypothesize that during aging macrophage polarization towards M2 is impaired, which in turn drives chronic inflammation and disturbs tissue maintenance. By combining state-of-the art lipidomic and proteomic profiling we aim to uncover new molecular targets for pharmaceutical interventions to improve therapeutic strategies for elderly patients with chronic inflammatory diseases.

Project description:Fibroblasts have only recently been identified as important effector cells in inflammation. In this study, human dermal fibroblasts were inflammatory stimulated with interleukin-1beta and comprehensively analysed with respect to proteins, eicosanoids and metabolites. For eicosadomics, we have established a data-dependent shotgun analysis method capable of identifying inflammation-regulated lipids of yet unknown function. Several classical inflammatory agonists were found induced, including PGA2, PGB2, PGE2 and TXB2, but also modulators such as PGA3 and PGE3, while 8-HETE and several HODE family members remained unaffected. Using targeted metabolomics, several acylcarnithins, phosphatitylcholins and sphingomyelins were found significantly downregulated. Proteome profiling with orbitrap-MS demonstrated the strong induction of several chemokines, metalloproteinases and other effector molecules. Treatment of stimulated fibroblasts with dexamethasone almost completely abrogated the formation of all inflammation-induced eicosanoids and restored levels of acylcarnithins back to normal. As expected, the secretion of IL-6, MMP1, MMP3, CXCL2 and CXCL3 was strongly down-regulated. However, instead of counter-regulating, dexamethasone further enhanced consequences of inflammatory stimulation with respect to CXCL1, CXCL6, complement C3 as well as sphingomyelins. Shotgun secretome data were confirmed by targeted analysis with triple-quadrupol-MS. These molecules have been described to be involved in chronic inflammation. In peripheral blood mononuclear cells, actually dexamethasone successfully downregulated the formation of all detectable inflammation mediators. The present data suggest that successful pharmacological abrogation of the formation of lipid inflammatory mediators in fibroblasts may not suffice to suppress the release of several other powerful inflammatory mediators which we thus understand to be capable of establishing chronic inflammation states.

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Organ-specific inflammaging in mice

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