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Local proliferation of macrophages in adipose tissue during obesity-induced inflammation

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Abstract

Aims/hypothesis

Obesity is frequently associated with low-grade inflammation of adipose tissue (AT), and the increase in adipose tissue macrophages (ATMs) is linked to an increased risk of type 2 diabetes. Macrophages have been regarded as post-mitotic, but recent observations have challenged this view. In this study, we tested the hypothesis that macrophages proliferate within AT in diet-induced obesity in mice and humans.

Methods

We studied the expression of proliferation markers by immunofluorescence, PCR and flow cytometry in three different models of mouse obesity as well as in humans (n = 239). The cell fate of dividing macrophages was assessed by live imaging of AT explants.

Results

We show that ATMs undergo mitosis within AT, predominantly within crown-like structures (CLS). We found a time-dependent increase in ATM proliferation when mice were fed a high-fat diet. Upregulation of CD206 and CD301 in proliferating ATMs indicated preferential M2 polarisation. Live imaging within AT explants from mice revealed that macrophages emigrate out of the CLS to become resident in the interstitium. In humans, we confirmed the increased expression of proliferation markers of CD68+ macrophages in CLS and demonstrated a higher mRNA expression of the proliferation marker Ki67 in AT from obese patients.

Conclusions/interpretation

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.

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Abbreviations

AT:

Adipose tissue

ATMs:

Adipose tissue macrophages

CCR2:

C-C motif chemokine receptor 2

CLS:

Crown-like structures

CSF1R:

Colony-stimulating factor 1 receptor

eGFP:

Enhanced green fluorescent protein

EWAT:

Epididymal white adipose tissue

HFD:

High-fat diet

MCP-1:

Monocyte chemoattractant protein 1

PBST:

PBS with 0.3% Triton-X

PCNA:

Proliferating cell nuclear antigen

SWAT:

Subcutaneous white adipose tissue

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Acknowledgements

We are grateful for the excellent technical assistance of C. Hobusch, A. Ehrlich and C. Merkwitz. We also thank M. Krüger for helpful discussions and K. Jäger and A. Lösche from the FACS core unit (all Leipzig University).

Funding

This work was in part supported by the Kompetenznetz Adipositas (Competence Network for Obesity) funded by the Federal Ministry of Education and Research (German Obesity Biomaterial Bank; FKZ 01GI1128 and FKZ 01EO1001), a grant from the Deutsche Forschungsgemeinschaft DFG-SFB 1052/1: ‘Obesity mechanisms’ (projects A04, B01, B04) and by the Helmholtz Alliance ‘Imaging and Curing Environmental Metabolic Disease’ through the Initiative and Networking Fund of the Helmholtz Association. MG received a start-up grant from the Medical Faculty of Leipzig University.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Contribution statement

JH planned and performed morphological analyses of murine and human AT samples. UW and JH analysed ATMs by flow cytometry. MG and JE designed, performed and evaluated live imaging experiments of AT explants. KI planned, established and analysed murine macrophage cultures. NK and MB analysed gene expression of human AT samples. IB and MG designed the study. JH and MG wrote the paper. All authors participated in drafting and revising this article. All authors approved the final version of the manuscript.

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Authors and Affiliations

  1. Institute of Anatomy, Leipzig University, Liebigstraße 13, 04103, Leipzig, Germany

    Julia Haase, Ulrike Weyer, Kerstin Immig, Ingo Bechmann & Martin Gericke

  2. IFB Adiposity Disease, Junior Research Group 2, Leipzig University, Leipzig, Germany

    Nora Klöting

  3. Department of Internal Medicine III, Leipzig University, Leipzig, Germany

    Matthias Blüher

  4. Carl-Ludwig Institute of Physiology, Leipzig University, Leipzig, Germany

    Jens Eilers

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  1. Julia Haase
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Correspondence to Martin Gericke.

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ESM Fig. 1

ATMs incorporate BrdU as a sign of cell replication. (A) Representative FACS plots are shown for chow controls (left) and mice after 10 weeks of HFD (right). F4/80+ ATMs were analyzed for BrdU incorporation (S phase) and the DNA content, measured by 7-AAD. Thus, ATMs in G1 phase (single DNA content) and G2 phase (double DNA content) can be distinguished. (B) Bar graph summarizing the percentage of resting (G0/G1) and proliferating (S/G2) ATMs after 1, 4 and 10 weeks of HFD. (PDF 97 kb)

ESM Fig. 2

ATMs proliferate in AT of ob/ob mice. (A-C) Triple immunofluorescence staining of the macrophage marker F4/80 (red), the proliferation marker Ki67 (green) and DAPI (blue) is shown. (A and B) Representative images of either ob/ob (n = 4) or ob/+ mice (n = 4). (C) Higher magnification and single color channels of the area depicted in (A) reveal a co-staining. Scale bar represents 20 μm in (A) and (B) or 10 μm in (C). (D) Quantification of Ki67+ ATMs in ob/+ and ob/ob animals. Data are shown for subcutaneous (SWAT) and epididymal (EWAT) AT. (E) Percentage of ATMs located within CLS for ob/+ and ob/ob mice (n.d. = not detected). (F) Percentage of Ki67+ ATMs in ob/ob mice not associated to CLS (interstitium) or associated to CLS (CLS). Data are represented as means ± SEM and tested for statistical significance by the Student-Newman-Keuls multiple comparison test. *** p ≤ 0.001 (PDF 52 kb)

ESM Fig. 3

ATMs proliferate in AT of db/db mice. (A-C) Triple immunofluorescence staining of the macrophage marker F4/80 (red), the proliferation marker Ki67 (green) and DAPI (blue) is shown. (A and B) Representative images of either db/db (n = 4) or db/+ mice (n = 3). (C) Higher magnification and single color channels of the area delineated in (A) reveal Ki67+ ATMs. Scale bar represents 20 μm in (A) and (B) or 10 μm in (C). (D) Bar graph summarizing the percentage of Ki67+ ATMs in db/+ and db/db mice. Data are shown for subcutaneous (SWAT) and epididymal (EWAT) AT. (E) Percentage of ATMs located within CLS for db/+ and db/db mice (n.d. = not detected). (F) Percentage of Ki67+ ATMs of db/db mice not associated to CLS (interstitium) or within CLS (CLS). Data are represented as means ± SEM and tested for statistical significance by the Student-Newman-Keuls multiple comparison test. ** p ≤ 0.01 and *** p ≤ 0.001 (PDF 63 kb)

ESM Video 1

ATMs migrate out of the CLS, rather than towards the CLS. Live-imaging of living AT explants was performed by using MacGreen mice (CSF-1R eGFP) to visualize ATMs in situ. Adipocytes were stained using Bodipy598 (blue), whereas ATMs are shown in green. Time is presented in h:min. (AVI 600 kb)

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Haase, J., Weyer, U., Immig, K. et al. Local proliferation of macrophages in adipose tissue during obesity-induced inflammation. Diabetologia 57, 562–571 (2014). https://doi.org/10.1007/s00125-013-3139-y

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