Hassnain Waqas SF et al. (2017), Adipose tissue macrophages develop from bone ma...

XB-ART-53796

J Leukoc Biol 2017 Sep 01;1023:845-855. doi: 10.1189/jlb.1A0317-082RR.

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Adipose tissue macrophages develop from bone marrow-independent progenitors in Xenopus laevis and mouse.

Hassnain Waqas SF , Noble A , Hoang AC , Ampem G , Popp M , Strauß S , Guille M , Röszer T .

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ATMs have a metabolic impact in mammals as they contribute to metabolically harmful AT inflammation. The control of the ATM number may have therapeutic potential; however, information on ATM ontogeny is scarce. Whereas it is thought that ATMs develop from circulating monocytes, various tissue-resident Mϕs are capable of self-renewal and develop from BM-independent progenitors without a monocyte intermediate. Here, we show that amphibian AT contains self-renewing ATMs that populate the AT before the establishment of BM hematopoiesis. Xenopus ATMs develop from progenitors of aVBI. In the mouse, a significant amount of ATM develops from the yolk sac, the mammalian equivalent of aVBI. In summary, this study provides evidence for a prenatal origin of ATMs and shows that the study of amphibian ATMs can enhance the understanding of the role of the prenatal environment in ATM development.

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Species referenced: Xenopus laevis
Genes referenced: atm itgam npff npffr1.1 ptprc rnf128 tal1

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	Figure 1. AT Mϕs in X. laevis.(A) Anatomy of the visceral fat depot. Scale bar, 1 cm. (B) Histologic assessment of the fat body by hematoxylin and eosin staining. Scale bar, 100 μm. (C) Enzyme histochemistry and ultrastructure of the fat body stroma, and quantification of ATMs. ACP- and peroxidase (POD)-positive cells in the fat body (top). Scale bar, 50 μm. TEM image of the fat body (bottom left). Arrowheads indicate ATMs (leukocytes with abundant endocytic vesicles). Scale bar, 1 μm. ATM content and percentage of phagocytosing (phag+) cells in the stroma (bottom right). The raw data set is available at the Flow Repository under accession number FR-FCM-ZYZF. (D) FACS analysis of SCs. For TEM images of the sorted RBCs, ATMs, and Lym, and for additional details of the FACS analysis, see Supplemental Fig. 1A–D. (E–G) Identification of phagocytosing cells in AT stroma. Isolated SCs were incubated in vitro with fluorescent latex beads for 4 h. (H) Time-lapse sequence of a phagocytosing ATM. The entire time lapse sequence is shown as Supplemental Video 1. Scale bar, 25 μm. (I) ATMs with ingested fluorescent latex beads (arrows). Scale bar, 15 μm. (J) TEM images of ATMs. Scale bar, 1 μm. Additional TEM images of ATMs are shown in Supplemental Fig. 1A. ac = adipocyte, fb = fat body; FSC = forward scatter, lp = lamellipodia, Lym = lymphocytes, nc = nucleus, ov, ovary; phag− = cells without phagocytosed beads, phag+ = cells with phagocytosed beads, SSC = side scatter, st = stroma, ves = blood vessel, vs = vesicles.
	Figure 2. Self-renewal of ATMs in X. laevis.(A) Isolated SCs were cultured in vitro and labeled with BrdU (indicated in red; n = 6). In the control experiment, cells were incubated with vehicle only (indicated in gray). Presence of BrdU+ cells was detected by FACS. The raw data set is available in the Flow Repository under accession number FR-FCM-ZYZG. (B) Characterization of BrdU+ cells with FACS. (C) Amount of BrdU+ ATMs after 2–4 h of incubation with BrdU. Histogram shows BrdU labeling after 4 h (n = 3). (D) SCs were cultured in vitro in soft agar. Colonies formed by SCs were detected after 24, 48, and 72 h. Arrow denotes cell-cell attachment site. Scale bar, 50 μm. (E) TEM images of SC colonies formed in soft agar. Representative images (E1-E3). Scale bar, 0.5 μm. cp = cytoplasm, FSC = forward scatter, Lym = lymphocytes, mt = mitochondria, nc = nucleus, SSC = side scatter.
	Figure 3. Development of ATMs in X. laevis.(A) Relative transcription of the hematopoietic marker scl in SCs of the fat bodies and in various hematopoietic tissues (n = 6; *P < 0.05, Student’s 2-tailed, unpaired t test). (B) Analysis of scl:eGFP+ cells in adult fat bodies using FACS and histology (n = 3). Arrows show scl:eGFP+ cells. Scale bar, 50 μm. Additional details are shown in Supplemental Fig. 2. (C) Relative transcription of the Mϕ precursor marker, lurp, in SCs of the adult fat bodies and in various hematopoietic tissues (n = 6). (D) Analysis of lurp:eGFP+ cells in adult fat bodies using FACS and histology (n = 3). Arrows show lurp:eGFP+ cells. Scale bar, 50 μm. Additional details are shown in Supplemental Fig. 2. (E1) Presence of lurp:eGFP+ cells in developing fat bodies. Arrows show lurp:eGFP+ cells. Scale bars, 1 cm (in macroscopic images showing developing tadpoles) and 50 μm (for the histology images). (E2) FACS analysis of lurp:eGFP+ ATMs at various developmental stages (n = 5 each stage). The data set is available in the Flow Repository under accession number FR-FCM-ZYZE. (F) Anatomy of the fat body in A. mexicanum. Scale bar, 1 cm. (G) Hematoxylin and eosin (H&E)–stained section and ACP enzyme histochemistry of the fat body. Arrow shows an ACP+ cell. Scale bar, 50 μm. ac = adipocyte, fb = fat body, FSC = forward scatter, st = stroma, ov = ovary.
	Figure 4. Homology of Xenopus and mouse ATM development.(A) FACS analysis of mouse SCs that were isolated from iWAT at postnatal day (PD) 7. Representative of 3 independent assays. Details of FACS analysis of ATMs have been described previously [19]. (B) TEM images of sorted cells of the iWAT stroma at PD7; also see Supplemental Fig. 4. Scale bar, 10 μm. (C) Identification of ATMs on the basis of CD11b, F4/80 antigen, and lysosomal protein Mac-3/Lamp-2 expression. Cells were isolated from iWAT at PD7. (D and E) Characterization of ATMs of the iWAT at PD7. (F) Immunohistochemistry against CX3CR1 in iWAT at PD7. F-Actin was labeled with fluorescent phalloidin. (G) Percentage of CX3CR1+ ATMs in the iWAT at PD7 and PD56. (H) Scheme for labeling YS-derived Mϕs in the mouse. (I) Percentage of YS-derived (CX3CR1-eGFP+) ATMs in wild-type (WT) and transgenic (TG) iWAT at PD7 (Flow Repository accession number FR-FCM-ZYZW). (J) Percentage of Ki-67+ ATMs in iWAT at PD7 and PD56, pooled data from 3 assays, and amount of CD45.2+ leukocytes in BM and blood and epididymal WAT ATMs, 20 wk after BM transplantation (n = 3; Flow Repository accession number FR-FCM-ZYZW). (K and L) Scheme of the proposed homology of ATM development in Xenopus and mouse. FSC, forward scatter; iMC = immature myeloid cell, lp = lamellipoda, nc = nucleus, ns = non-significant, POD = peroxidase, SSC = side scatter, vs = vesicles. ** P<0.01, ANOVA with Dunnett’s post-hoc test.
	Figure 5. Control of ATM number and activation state by gpr147 and NPFF.(A) Relative transcription of gpr147 mRNA in ATMs, adipocytes (ACs), and various tissues (n = 4). Transcript levels are normalized to ATMs. Insert shows predicted primary structure of Xenopus gpr147 using LOMETS structure prediction software [84]. (B) Transcription of lurp in wild-type (WT) and gpr147-KO fat body (n = 5). (C) ACP+ cells (arrowhead) in fat body of WT and gpr147-KO adult frog. Scale bar, 50 μm. (D) Number of ATMs that were isolated from WT and gpr147-KO fat bodies of adult frogs (n = 5). (E) Representative images of SCs of a WT frog cultured in soft agar and treated with vehicle or 0.5 nM NPFF. Colonies were photographed at day 3 of treatment. (F) Size of vehicle- or NPFF-treated colonies (n > 80 each assay), pooled data of 3 assays. (G) Relative transcription of Mϕ activation genes in ATMs of WT and gpr147-KO frogs (n = 5). (H) Xenopus ATMs were treated with vehicle or 1 nM NPFF for 1 h in vitro and relative transcription of Mϕ activation genes was measured (n = 3). (I) Relative transcription of rnf128 in WT and npffr1-KO ATMs (n = 5), and in ATMs treated in vitro with vehicle or 1 nM NPFF for 1 h (n = 3). (J) Relative transcription of Mϕ activation genes in ATMs of in vitro cultured Xenopus ATMs, treated with vehicle, 1 nM NPFF, pI:pC, or combination of pI:pC and NPFF for 4 h (n = 3). ac = adipocyte. P < 0.05; P < 0.01; **P < 0.001, Student’s t test (B–I), or 1-way ANOVA with Dunnett’s post hoc test (A and J).

References [+] :

Ablamunits, Reduced adiposity in ob/ob mice following total body irradiation and bone marrow transplantation. 2007, Pubmed