Supplementary MaterialsSupplementary Information 41467_2020_16758_MOESM1_ESM. remain incompletely understood. Here we display profound beiging inside a genetic mouse model lacking the transcriptional repressor Krppel-like element 3 (KLF3). Bone marrow transplants from these animals confer the beige phenotype on crazy type recipients. Analysis of the cellular and molecular changes reveal an accumulation of eosinophils in adipose tissue. We examine the transcriptomic profile of adipose-resident eosinophils and posit that KLF3 regulates adipose tissue function via transcriptional control of secreted molecules linked to beiging. Furthermore, we provide evidence that eosinophils may directly act on adipocytes to drive TLR3 beiging and spotlight the critical role of these little-understood immune cells in thermogenesis. mice and that bone marrow (BM) transplants from these mice confer the lean, beige phenotype on recipients. In the absence of KLF3, AT-resident eosinophils are more abundant and exhibit significant deregulation of important secreted molecules, including meteorin-like and IL-33, both of which influence beiging5,14C16. We also report that co-culture of eosinophils with primary adipocytes increases thermogenic gene expression. These findings identify KLF3 as an important regulator of AT eosinophil gene expression and function, advancing our understanding of how these little-understood immune cells may lead to improved strategies for therapeutically driving energy expenditure. Results Reduced adiposity and enhanced beiging in animals housed at room heat (22?C) showed that mice exhibit reduced total fat mass compared Z-DEVD-FMK reversible enzyme inhibition to WT littermates (Fig.?1a and Supplementary Fig.?1a), in addition to differences in lean body mass, which constitute their reduced body weight (Supplementary Fig.?1b). This is reflected in the reduced size of white AT depots in mice seen previously12 (Fig.?1b and Supplementary Fig.?1c). Visual examination of subcutaneous (subcut) AT depots, the depots most prone to beiging17, revealed a browner complexion and smaller size in mice (Fig.?1c). Furthermore, H&E staining revealed that in the absence of KLF3, adipose Z-DEVD-FMK reversible enzyme inhibition cellular architecture is usually notably altered, with enrichment of multilocular adipocytes evident that was not seen in the subcut AT of WT mice (Supplementary Fig.?2a,?b). These observations also confirm the previous finding that mice have smaller-sized adipocytes12,13. Given that thermogenic energy expenditure via activation of beige AT may influence adiposity18C20, we examined the expression of archetypal thermogenic genes. We observed upregulation of numerous thermogenic genes in the subcut AT of mice C most notably and the beige-specific marker21 (Fig.?1d). We next investigated the levels of mitochondrial proteins by Western blotting of whole-cell extracts (WCE) from WT and subcut AT. Uncoupling protein 1 (UCP1) protein levels were higher in the subcut AT of mice (Fig.?1e), as were mitochondrial oxidative phosphorylation (oxphos) complexes ICV (Fig.?1f). We also observed increased levels of the mitochondrial outer membrane protein voltage-dependent anion channel (VDAC) in subcut AT (Fig.?1g), suggesting higher mitochondrial number. Levels of multiple thermogenic genes were also increased in the gonadal AT of mice (Supplementary Fig.?1d), as were UCP1 and mitochondrial oxphos proteins (Supplementary Fig.?1e, f). Several genes were modestly increased in interscapular brown AT of mice while beige-specific markers1 and were undetectable (Supplementary Fig.?1g). UCP1 protein content was mildly decreased in brown AT (Supplementary Fig.?1h, i). While this suggests that brown AT is unlikely to play a major role in the thermogenic phenotype of mice, we cannot wholly rule out its contribution given the presence of UCP1-impartial thermogenic mechanisms in beige and brown excess fat22C25. Together, these results show that mice exhibit reduced fat mass that may result from enhanced AT beiging, as demonstrated by the widespread de-repression of thermogenic genes and mitochondrial proteins. Open in a separate windows Fig. 1 Reduced adiposity and enhanced beiging in mice.a Lean and fat body mass composition (%) of WT ((AT depots were recorded as a percentage of total body weight (subcut AT pads showing relative size and complexion. d mRNA levels of thermogenic genes were assessed by qPCR in WT and subcut AT (rRNA levels and the mean WT value for each gene was set to 1 1. e UCP1 protein expression was measured in WT and subcut AT by western blotting (subcut AT WCE was assessed by western blotting (subcut AT (assessments were performed where *mice to ambient temperatures of Z-DEVD-FMK reversible enzyme inhibition 4?C or 30?C (Fig.?2a). No statistically significant difference was seen in the body temperatures of WT and mice during cold exposure (Fig.?2b). As expected, there was.