Local proliferation contributes to the increase in M2 macrophages in AT. Our data confirm CLS as the primary site of proliferation and a new source of ATMs and support a model of different recruitment mechanisms for classically activated (M1) and alternatively activated (M2) macrophages in obesity.
Obesity is associated with chronic low-grade inflammation of adipose tissue (AT) and an increase of AT macrophages (ATMs) that is linked to the onset of type 2 diabetes. We have recently shown that focal sites of inflammation around dying adipocytes, so-called crown-like structures, exhibit a unique microenvironment for macrophage proliferation. Interestingly, locally proliferating macrophages were not classically activated (M1), but they exhibited a rather alternatively activated (M2) immune phenotype. In this study, we established organotypic cell cultures of AT explants to study the impact of cytokine treatment on local ATM proliferation, without the bias of early monocyte recruitment. We show that exposure of AT to Th2 cytokines, such as IL-4, IL-13, and GM-CSF, stimulates ATM proliferation, whereas Th1 cytokines, such as TNF-α, inhibit local ATM proliferation. Furthermore, AT from obese mice exhibits an increased sensitivity to IL-4 stimulation, indicated by an increased phosphorylation of STAT6. In line with this, gene expression of the IL-4 receptor () and its ligand IL-13 are elevated in AT of obese C57BL/6 mice. Most importantly, expression and susceptibility to IL-4 or IL-13 treatment depend on IL-6 signaling, which seems to be the underlying mechanism of local ATM proliferation in obesity. We conclude that IL-6 acts as a Th2 cytokine in obesity by stimulating M2 polarization and local ATM proliferation, presumably due to upregulation of the IL-4 receptor α.
SUMMARYFunctional analyses of the pl0 gene promoter from the Autographa californica nuclear polyhedrosis virus (AcNPV) were performed by progressively deleting the 230 nucleotides upstream from the pl0 coding sequences towards the ATG codon. Truncated promoter sequences retaining the full 5' non-coding leader of pl0 were inserted in front of the chloramphenicol acetyltransferase (CAT) gene, and promoter activity in transfected AcNPV-infected cells was measured using the transient CAT expression assay. The removal of sequences to a position 101 nucleotides upstream from the pl0 ATG did not affect the level of CAT expression. Deletion of a further 13 nucleotides reduced CAT expression by three-to fourfold, but the removal of three more nucleotides, which deleted most of the baculovirus very late gene transcription consensus sequence, almost completely abolished activity. The removal of the TATA motif had no effect on the level of transient expression. We conclude that a sequence of about 101 nucleotides upstream from the ATG codon of p l0 is sufficient for high level promoter activity in this transient system.
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