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. 2020 Nov 5;9:e55676. doi: 10.7554/eLife.55676

Figure 2. miR-17~92 family miRNAs maintain adipose tissue macrophage (ATM) homeostasis.

(A) Glucose tolerance test in WT and TKO mice (left panel); area under curve (AUC) of left panel (right panel), n = 6 per group. (B) qPCR analysis of Tnf and Il10 mRNA in visceral adipose tissue of WT and TKO mice. (C and D) Flow cytometry of ATM population in WT and TKO mice. (C) A representative figure showing the ATM population of CD45+ cells; (D) cumulative quantification of ATM population as in (C). (E and F) ATMs were sorted from WT and TKO mice fed with regular chow diet, RNA samples of ATMs were extracted and pooled from two mice, and genome-wide RNA profiling analyses were performed. (E) Gene ontology (GO) analysis of WT and TKO ATM RNA-seq datasets showing the enriched GO terms in TKO ATMs; (F) RNA-seq analysis showing RNA expression in TKO ATMs versus those in WT cells. RNAs upregulated in TKO ATMs were colored red, whereas RNAs downregulated were colored blue, gene Tnf was pointed out and colored orange, and gene Il10 was pointed out and colored green. Top 10 upregulated genes (Ighg2b, Gm11843, Dcdc2a, Mmp7, Slc38a5, Tenm2, Cdh16, Ptn, Ighv1-42, and Nanp) were colored yellow and top 10 downregulated genes (Csf2ra, Zfp125, Cxcl5, Ighv14-3, Igkv4-59, Igkv6-20, Igkv16-104, Fth-ps2, Igkv4-91, and B230303A05Rik) were colored bright blue. (G and H) qPCR analysis of Tnf (G) and Il10 (H) mRNA in ATMs of WT and TKO mice. Results were shown as relative expression normalized to those expressions in one WT mice of each experiment. NS, not significant (p>0.05); *p<0.05, ***p<0.001, and ****p<0.0001 (unpaired Student’s t-test). Data are representative of three independent experiments (C) or are pooled from two (A), three (B and D), or five (G and H) independent experiments (mean ± sem).

Figure 2.

Figure 2—figure supplement 1. miR-17~92 family miRNAs inhibit TNF and promote IL-10 expression in adipose tissue.

Figure 2—figure supplement 1.

(A and B) Protein levels of TNF (A) and IL-10 (B) in serum of WT and TKO mice measured by ELISA. (C and D) qPCR analysis of Tnf (C) and Il10 (D) mRNA with additional primer pairs in WT and TKO adipose tissues. *p<0.05, **p<0.01 (unpaired Student’s t-test). Data are pooled from three (A–D) independent experiments (mean ± s.e.m.).
Figure 2—figure supplement 2. The miR-17~92 family miRNAs are efficiently deleted in TKO adipose tissue macrophages (ATMs).

Figure 2—figure supplement 2.

(A and B) Flow cytometry analysis of RFP expression in ATMs (A) and peritoneal macrophages (B, positive control) from Lyz2-Cre and Lyz2-RFP mice. (C–E) qPCR analysis of various mature miRNAs (horizontal axes) in the miR-17~92 cluster (C), miR-106a~363 cluster (D), and miR-106b~25 cluster (E) in Lyz2-Cre (WT) andTKO ATMs; results are presented as relative expression normalized to the control small RNA U6. Data are representative of two independent experiments (mean + s.d.).
Figure 2—figure supplement 3. The adipose tissue macrophage (ATM) populations are not changed in TKO mice.

Figure 2—figure supplement 3.

(A–E) Flow cytometry analysis of CD11c (A), CD64 (B), CX3CR1 (C), MerTK (D), and Ly6C (E) expression in ATMs from Lyz2-Cre (WT) and TKO mice. Data are representative of two independent experiments.
Figure 2—figure supplement 4. Adipose tissue macrophages (ATMs) in TKO mice are more inflammatory.

Figure 2—figure supplement 4.

(A and B) WT and TKO mice were fed with regular chow diet or high-fat diet (HFD) for 8 weeks; ATMs were sorted for a genome-wide RNA profiling analysis. (A) Gene ontology analysis of RNA-seq datasets of ATMs from WT mice fed with chow diet or HFD. (B) Inflammatory score of WT and TKO mice fed with chow diet or HFD. Inflammatory score = sum of log (FPKM+1) of representative pro-inflammatory genes (Tnf, Il6, Cxcl1, Ccl2, and Ccl8) − sum of log (FPKM+1) of representative anti-inflammatory genes (Il10, Il4, Il5, and Il13). (C and D) qPCR analysis of Tnf (C) and Il10 (D) mRNA with an additional set of primers in WT and TKO ATMs. *p<0.05 (unpaired Student’s t-test). Data are representative of one (A and B) or are pooled from two (C and D) independent experiments (mean ± s.e.m.).