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Alternatively activated macrophages (AAMs) contribute to the resolution of inflammation and tissue repair. However, molecular pathways that govern their differentiation have remained incompletely understood. Here, we show that uncoupling protein-2-mediated mitochondrial reprogramming and the transcription factor GATA3 specifically controlled the differentiation of pro-resolving AAMs in response to the alarmin IL-33. In macrophages, IL-33 sequentially triggered early expression of pro-inflammatory genes and subsequent differentiation into AAMs. Global analysis of underlying signaling events revealed that IL-33 induced a rapid metabolic rewiring of macrophages that involved uncoupling of the respiratory chain and increased production of the metabolite itaconate, which subsequently triggered a GATA3-mediated AAM polarization. Conditional deletion of GATA3 in mononuclear phagocytes accordingly abrogated IL-33-induced differentiation of AAMs and tissue repair upon muscle injury. Our data thus identify an IL-4-independent and GATA3-dependent pathway in mononuclear phagocytes that results from mitochondrial rewiring and controls macrophage plasticity and the resolution of inflammation. [Display omitted] • IL-33 induces rapid mitochondrial rewiring in macrophages • Uncoupling of the respiratory chain promotes IL-33-induced AAM differentiation • GATA3 links macrophage metabolism and differentiation in response to IL-33 • GATA3 orchestrates the differentiation of pro-resolving macrophages in vivo Alternatively activated macrophages (AAMs) promote the resolution of inflammation and tissue repair. Faas et al. show that the alarmin IL-33 promotes a mitochondrial rewiring of macrophages and the consecutive activation of the transcription factor GATA3, which orchestrates the IL-4-independent differentiation of AAMs and the resolution of inflammation upon tissue injury. [ABSTRACT FROM AUTHOR]

Summary: Noninflammatory clearance of apoptotic cells (ACs) is crucial to maintain self-tolerance. Here, we have reported a role for the enzyme 12/15-lipoxygenase (12/15-LO) as a central factor governing the sorting of ACs into differentially activated monocyte subpopulations. During inflammation, uptake of ACs was confined to a population of 12/15-LO-expressing, alternatively activated resident macrophages (resMΦ), which blocked uptake of ACs into freshly recruited inflammatory Ly6Chi monocytes in a 12/15-LO-dependent manner. ResMΦ exposed 12/15-LO-derived oxidation products of phosphatidylethanolamine (oxPE) on their plasma membranes and thereby generated a sink for distinct soluble receptors for ACs such as milk fat globule-EGF factor 8, which were essential for the uptake of ACs into inflammatory monocytes. Loss of 12/15-LO activity, in turn, resulted in an aberrant phagocytosis of ACs by inflammatory monocytes, subsequent antigen presentation of AC-derived antigens, and a lupus-like autoimmune disease. Our data reveal an unexpected key role for enzymatic lipid oxidation during the maintenance of self-tolerance. [ABSTRACT FROM AUTHOR]

Tissue-resident macrophages exhibit specialized phenotypes dependent on their in vivo physiological niche. Investigation of their function often relies upon complex whole mouse transgenic studies. While some appropriate lineage-associated promoters exist, there are no options for tissue-specific targeting of macrophages. We have developed full protocols for in vivo productive infection (defined by stable transgene expression) of tissue-resident macrophages with lentiviral vectors, enabling RNA and protein overexpression, including expression of small RNA species such as shRNA, to knock down and modulate gene expression. These approaches allow robust infection of peritoneal tissue-resident macrophages without significant infection of other cell populations. They permit rapid functional study of macrophages in homeostatic and inflammatory settings, such as thioglycolate-induced peritonitis, while maintaining the cells in their physiological context. Here we provide detailed protocols for the whole workflow: viral production, purification, and quality control; safety considerations for administration of the virus to mice; and assessment of in vivo transduction efficiency and the low background levels of inflammation induced by the virus. In summary, we present a quick and accessible protocol for the rapid assessment of gene function in peritoneal tissue-resident macrophages in vivo ., (© 2019 The Authors.)

The effect of metabolic stress on the bone marrow microenvironment is poorly defined. We show that high-fat diet (HFD) decreased long-term Lin(-)Sca-1(+)c-Kit(+) (LSK) stem cells and shifted lymphoid to myeloid cell differentiation. Bone marrow niche function was impaired after HFD as shown by poor reconstitution of hematopoietic stem cells. HFD led to robust activation of PPARγ2, which impaired osteoblastogenesis while enhancing bone marrow adipogenesis. At the same time, expression of genes such as Jag-1, SDF-1, and IL-7 forming the bone marrow niche was highly suppressed after HFD. Moreover, structural changes of microbiota were associated to HFD-induced bone marrow changes. Antibiotic treatment partially rescued HFD-mediated effects on the bone marrow niche, while transplantation of stools from HFD mice could transfer the effect to normal mice. These findings show that metabolic stress affects the bone marrow niche by alterations of gut microbiota and osteoblast-adipocyte homeostasis., (Copyright © 2015 Elsevier Inc. All rights reserved.)