Project description:Chronic low-grade inflammation is a hallmark of aging, but its etiology is not well understood. Mitochondrial dysfunction has been linked to both cellular senescence and age-related inflammation or inflammaging but fundamental mechanisms underlying these links are unclear. We analyzed age-related gene expression in different human tissues and discovered that in blood, the mRNA expression of Mitochondrial Calcium Uniporter (MCU) and its regulatory subunit MICU1 correlated inversely with age. MCU is the pore-forming subunit of a Ca2+-selective ion channel complex residing in the mitochondrial inner membrane and is the main conduit for Ca2+-influx into the mitochondrial matrix. Based on these findings we tested the simple hypothesis that mitochondrial Ca2+ uptake capacity of macrophages decreases with age. We found a significant reduction in the mitochondrial Ca2+-uptake capacity of macrophages derived from aged (80-90 weeks) mice. Notably, these macrophages display amplified cytosolic Ca2+ elevations and increased inflammatory output. To test the salience of mitochondrial calcium uptake in regulating macrophage inflammatory output, we deleted Mcu selectively in macrophages. The Mcu-/- macrophages of young mice (<25 weeks) express more inflammatory genes at baseline and show a hyper-inflammatory response when stimulated. We show that reduced mitochondrial Ca2+ amplifies cytosolic Ca2+ oscillations and potentiates downstream NFkB activation, which is central to inflammation. The regulation of inflammatory response by mitochondrial Ca2+ uptake is also seen readily in human macrophages. The siRNA-mediated knockdown of MCU in human blood derived macrophages recapitulates the phenomenon – the mitochondrial Ca2+-uptake is greatly reduced, cytosolic Ca2+ elevations are more pronounced, and the macrophages are hyper-inflammatory. Our findings pinpoint the MCU complex as a keystone molecular apparatus that links age-related changes in mitochondrial physiology to macrophage-mediated inflammation. Resident macrophages are intrinsic to every organ system and the steady erosion of their mitochondrial Ca2+-uptake capacity may play a germinal or exacerbating role in many age-related neurodegenerative, cardiometabolic, renal and musculoskeletal diseases that afflict us.

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.

			Action	DRS
	190616ko02NT_gLuc_LA_1.maxq.dat	Other
	190616ko02NT_gLuc_LA_1.maxq.txt	Txt
	190616ko02NT_gLuc_LA_1.raw	Raw
	190616ko03MD_gLuc_Lamp2C_1.maxq.dat	Other
	190616ko03MD_gLuc_Lamp2C_1.maxq.txt	Txt

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Discovery of phosphatidylserine-deficient small extracellular vesicle subpopulation with super long blood circulation

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