A method for long-term live imaging of tissue macrophages in adipose tissue explants

Abstract

Obesity is frequently associated with a chronic low-grade inflammation within adipose tissue (AT). Although classical signs of inflammation are missing in AT inflammation, there is a significant increase in macrophages and, to a lesser extent, other immune cells, such as T cells, B cells, mast cells, and neutrophils. The spatial and temporal activation of these cells as well as their accumulation in the AT seem to be tightly linked to so-called crown-like structures (CLS). CLS are accumulations of adipose tissue macrophages (ATMs) around dead adipocytes and are thought to reflect a scavenger response. At present, data on the life cycle of CLS are missing. To better understand the cellular events underlying AT inflammation, we developed an approach that allows long-term imaging of ATMs, adipocytes, and CLS within live AT explants. We tested three putative reporter mouse lines for myeloid cells in regard to their suitability for live imaging. Thereby, we identified ATMs from CSF1R-eGFP mice to exhibit the most robust expression of eGFP. AT explants from these mice allowed stable live imaging for more than 7 days without significant phototoxicity. Long-term imaging thus revealed the accumulation of ATMs around dying adipocytes, migration of ATMs within AT, and also the degradation of the lipid remnants of perishing adipocytes. The observed behavior of ATMs in the context of AT inflammation is in line with previous studies but for the first time provides data on the specific behavior of individual ATMs and on the life cycle of CLS with unprecedented spatiotemporal resolution.

Hedgehog signalling in myeloid cells impacts on body weight, adipose tissue inflammation and glucose metabolism

“…Live imaging of migration of ATMs in living tissue Staining of ATMs using AlexaFluor647-conjugated isolectin B4 (IB4) from Griffonia simplicifolia (ThermoFisher Scientific; Dreieich, Germany) and subsequent live imaging of adipose tissue explants were performed as described before in detail [28]. Images were obtained every 15 min and dynamic parameters of ATMs (5 movies per genotype) were validated in an unbiased, automated fashion using the Trackmate plugin from Fiji software 2.0 [29].…”

Properties and functions of adipose tissue macrophages in obesity

“…Although it is not fully resolved what triggers the activation of ATMs, there is evidence that adipocyte hypertrophy and local hypoxia, following energy imbalance, instigate ATM accumulation, 3,43,53 and a switch in the phenotype of ATMs from an anti-inflammatory state to a pro-inflammatory state. 21 Moreover, not only the accumulation of macrophages, but also an impairment in macrophage egress has been implicated in obesity-induced inflammation.…”

Unspecific DNA recombination in AdipoqCre-ERT2 – mediated knockout approaches in transgenic mice is sex-, age- and genotype-dependent

“…We used young, male and heterozygote Adipoq-CreER T2 xTDTO reporter mice for visualization of recombination in live AT after tamoxifen treatment using an established tissue culture model [14]. Briefly, after sacrifice, the rostral part of the epididymal white adipose tissue (EWAT) was dissected under sterile conditions and transferred to a PBSfilled culture dish (Life Technologies, Darmstadt, Germany).…”

“…However, TAM administration in living mice has also been suggested to affect adipocyte biology [11] and may even induce adipocyte death in vivo and in vitro [12,13]. Therefore, we aimed at establishing a Cre/loxP-based ex vivo system to visualize adipocytes and study the site of DNA recombination in real time using a live-imaging approach as described previously [14]. Further, we wanted to analyse the impact of TAM on mature adipocytes in living AT explants.…”