KLF10 Deficiency in CD4+ T Cells Triggers Obesity, Insulin Resistance, and Fatty Liver - PubMed (original) (raw)

. 2020 Dec 29;33(13):108550.

doi: 10.1016/j.celrep.2020.108550.

Shijia Wang 2, Chun Wu 2, Fang Fang 3, Stefan Haemmig 1, Brittany N Weber 1, Ceren O Aydogan 4, Yevgenia Tesmenitsky 1, Hassan Aliakbarian 5, John R Hawse 6, Malayannan Subramaniam 6, Lei Zhao 7, Peter T Sage 8, Ali Tavakkoli 9, Amanda Garza 10, Lydia Lynch 10, Alexander S Banks 11, Mark W Feinberg 12

Affiliations

KLF10 Deficiency in CD4+ T Cells Triggers Obesity, Insulin Resistance, and Fatty Liver

Akm Khyrul Wara et al. Cell Rep. 2020.

Abstract

CD4+ T cells regulate inflammation and metabolism in obesity. An imbalance of CD4+ T regulatory cells (Tregs) is critical in the development of insulin resistance and diabetes. Although cytokine control of this process is well understood, transcriptional regulation is not. KLF10, a member of the Kruppel-like transcription factor family, is an emerging regulator of immune cell function. We generated CD4+-T-cell-specific KLF10 knockout (TKO) mice and identified a predisposition to obesity, insulin resistance, and fatty liver due to defects of CD4+ Treg mobilization to liver and adipose tissue depots and decreased transforming growth factor β3 (TGF-β3) release in vitro and in vivo. Adoptive transfer of wild-type CD4+ Tregs fully rescued obesity, insulin resistance, and fatty liver. Mechanistically, TKO Tregs exhibit reduced mitochondrial respiration and glycolysis, phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR signaling, and consequently impaired chemotactic properties. Collectively, our study identifies CD4+ T cell KLF10 as an essential regulator of obesity and insulin resistance by altering Treg metabolism and mobilization.

Keywords: CD4(+) T cell; KLF10; PI3K-Akt-mTOR pathway; Treg; glycolysis; metabolic disorders; mitochondria; obesity; oxidative phosphorylation.

Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Interests The authors declare no competing interests.

Figures

Figure 1.

Figure 1.. CD4+ T Cells Exhibit Decreased KLF10 Expression in Obese Mice and Human Subjects

(A) KLF10 relative expression in splenic CD4+CD25− T cells (Teffs) and CD4+CD25+ T regulatory cells (Tregs) after 12 weeks of chow diet or HFD conditions (n = 6 per group). (B) KLF10 relative expression in peripheral blood CD4+CD25− T cells (Teffs) and CD4+CD25+ T regulatory cells (Tregs) after 12 weeks of chow diet or HFD conditions (n = 6 per group). (C) KLF10 relative expression in CD4+ Teffs and Tregs from peripheral blood of human subjects with BMI of <24 or BMI of >35. (D) KLF10 relative expression in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) after 12 weeks of chow diet or HFD conditions (n = 6 per group). (E) KLF10 relative expression in CD4+ Teffs and Tregs of SAT and VAT after 12 weeks of chow diet or HFD conditions (n = 6 per group). Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM.

Figure 2.

Figure 2.. CD4+ T Cell KLF10-Deficient (TKO) Mice Develop Insulin Resistance, Fatty Liver, and Adipose Inflammation with Reduced Tissue Treg Accumulation

(A) Body weights of WT and TKO mice over 12 weeks of HFD (n = 10 per group). (B) Body composition of WT and TKO mice after HFD for 12 weeks (n = 6 per group). (C and D) Glucose tolerance test (GTT) (C) and insulin tolerance test (ITT) (D) were performed on WT and TKO mice after 12 weeks of HFD (n = 10 per group). AUC, area under the curve. (E) Representative liver sections were stained with oil red O (ORO) (top panels) or hematoxylin and eosin (H&E) (middle panels) or immunostained against Mac2 for macrophages (bottom panels) (n = 10 per group; 5 random fields for each mouse; scale bars, 100 μm) (F and G) Representative sections of VAT and SAT immunostained against Mac2 (n = 10 per group; 5 random fields for each mouse; scale bars, 100 μm). (H–J) Quantification by flow cytometry of CD25 and Foxp3 expression in CD4+ T cells in liver (H), VAT (I), and SAT (J) of WT and TKO mice. Bar graphs show percentages of CD4+CD25+Foxp3+ Treg cells and CD4+CD25+Foxp3− T cells (n = 4 mice per group). (K–M) WT and TKO mice were placed on 4 weeks of HFD and assessed in metabolic cages. Energy expenditure (K) and energy expenditure regression plots correlated with total body weights are shown (L and M). Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. NS, non-significant. Results are reported as mean ± SEM. Related to Figures S1 and S2.

Figure 3.

Figure 3.. TKO Tregs Display Marked Defects in Mobilization In Vitro and In Vivo

(A) CD4+CD25− T cells from spleens of WT and TKO mice after 12 weeks of HFD were treated with anti-CD3 antibodies and TGF-β1 for differentiation into in vitro differentiated Tregs (iTregs). Percentage of WT and TKO CD4+CD25+Foxp3+ Tregs were measured by flow cytometry at the indicated time points (n = 6 per group). (B and C) CD4+CD25− T cells from spleens of WT and TKO mice after 12 weeks of HFD were activated by anti-CD3 antibodies for 24 h and subjected to qRT-PCR analysis (B) or ELISA from supernatants (C) for the indicated cytokines, chemokines, and growth factors (n = 5–9 per group). (D and E) Transwell migration study of CD4+CD25+ Tregs isolated from WT and TKO mice after 12 weeks of HFD. Cells were assessed for migration in the presence or absence of CCL19 (D) or CCL20 (E) (n = 3 per group). (F and G) Flow cytometry for CCR7 (F) or CCR6 (G) expression in WT and TKO Tregs (n = 6 per group). (H) Schematic of PKH26-labeled HFD WT and TKO Tregs adoptively transferred to HFD C57BL/6 mice. Flow cytometry shows percentage of PKH26-expressed cells in liver, VAT, and SAT of recipient mice (n = 6 per group). (I and J) Schematic of glucose uptake study of differentiated 3T3-L1 cells co-cultured with HFD WT and TKO iTreg supernatant (supe) (I). (J) Fluorescence intensity of 2-Deoxy-D-glucose (2-DG) uptake by differentiated 3T3-L1 cells co-cultured with supernatants of WT and TKO CD4+ Tregs in the presence or absence of insulin stimulation (n = 4 per group). (K and L) Schematic of glucose production study of mouse primary hepatocytes co-cultured with HFD WT and TKO iTreg supernatants (K). (L) Glucose production by mouse primary hepatocytes co-cultured with supernatants of HFD WT and TKO CD4+ Tregs (n = 6 per group). Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM. Related to Figures S3 and S4.

Figure 4.

Figure 4.. Adoptive Transfer of WT Tregs Fully Rescues Obesity, Insulin Resistance, and Fatty Liver in TKO Mice

(A) Schematic of adoptively transferred splenic WT and TKO CD4+ Tregs (500,000 cells/mouse per week) into WT and TKO recipient mice during 12 weeks of HFD (n = 6 per group). (B) Body weights of the four recipient mouse groups over 12 weeks: (1) WT mice + phosphate-buffered saline (PBS), (2) TKO mice + PBS, (3) TKO mice + WT Tregs, and (4) TKO mice + TKO Tregs (n = 6 per group). (C) Body composition of the four recipient mouse groups over 12 weeks: (1) WT mice + PBS, (2) TKO mice + PBS, (3) TKO mice + WT Tregs, and (4) TKO mice + TKO Tregs (n = 6 per group). (D and E) GTT (D) and ITT (E) were performed on the indicated recipient HFD mice after 12 weeks (n = 6 per group). (F) Representative liver sections were stained with ORO (n = 6 per group; 5 random fields per mouse; scale bars, 100 μm). (G and H) Representative sections of VAT and SAT were immunostained against Mac2 for macrophages (n = 6 per group; 5 random fields per mouse; scale bars, 100 μm). Bar graphs on the right (top) represent percentage of Mac2 staining as percentage of whole field; bar graphs on the right (bottom) represent counts of crown-like structures. Beneath the images, pie charts and bar graphs represent the percentages of small, medium, and large adipocytes in each group. (I) Plasma concentrations of metabolism-related hormones insulin, C-peptide, resistin, and leptin in the indicated groups after 12 weeks HFD (n = 6 per group). Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM. Related to Figure S4.

Figure 5.

Figure 5.. Signaling Pathway Analysis of KLF10 Deficiency in CD4+ T Cells

CD4+CD25+ Tregs and CD4+CD25− Teffs isolated from spleens of WT and TKO mice after 12 weeks of HFD mice were subjected to the following analyses. (A) Gene Ontology (GO) analyses of molecular and cellular functions of WT and TKO Tregs based on fold change (FC) of >1.5, p < 0.05, and RPKM of >1 genes (560 genes) by ingenuity pathway analysis (IPA) (n = 2–3 mice per group). (B) Signaling pathway analysis of WT and TKO Tregs based on full set of genes (48,440 genes) by gene set enrichment analysis (GSEA). (C) Heatmap of representative genes involved in oxidative phosphorylation, PI3K-Akt-mTOR signaling, TGF-beta signaling, and glycolysis in WT and TKO Tregs. (D) Representative signaling pathway network. Pink represents genes up-regulated in TKO compared to WT Tregs. Gray represents genes affected but without significant FCs. Green represents down-regulated genes in TKO Tregs. (E) Enrichment plots of WT versus TKO Tregs in oxidative phosphorylation (top) and PI3K-Akt-mTOR (bottom) signaling pathways. (F) Western blot analyses for p-Akt-ser473, total-AKT (T-AKT), p-ERK, total-ERK (T-ERK), p-mTOR, and total mTOR (T-mTOR) protein expression of Tregs from WT and TKO mice after 12 weeks of HFD (lysates pooled from n = 4 mice per group). (G) GO analyses of molecular and cellular functions of WT and TKO Teffs based on FC of >1.5, p < 0.05, and RPKM of >1 genes (560 genes) by IPA (n = 2 mice per group). (H) Signaling pathway analysis of WT and TKO Teffs based on full set of genes (48,440 genes) by GSEA. (I) Heatmap of representative genes involved in oxidative phosphorylation, PI3K-Akt-mTOR signaling, TGF-beta signaling, and glycolysis in WT and TKO Teffs. (J) Representative signaling pathway network. Pink represents genes up-regulated in TKO compared to WT Teffs. Gray represents genes affected but without significant FCs. Green represents genes down-regulated in TKO Teffs. (K) Enrichment plots of WT vs TKO Teffs in oxidative phosphorylation (top) and PI3K-Akt-mTOR (bottom) signaling pathways. (L) Western blot analyses for p-Akt-ser473, T-AKT, p-ERK, T-ERK, p-mTOR, and T-mTOR protein expression of Teffs from WT and TKO mice after 12 weeks of HFD (lysates pooled from n = 4 mice per group). Related to Figure S5.

Figure 6.

Figure 6.. TKO Tregs Exhibit Severe Defects in Oxidative Phosphorylation and Glycolysis

(A and B) Oxygen consumption rates (OCRs) of iTregs (A) or CD4+CD25− Teffs (B) from spleens of WT or TKO mice after 12 weeks of HFD as measured by Seahorse (n = 4 per group). (C and D) Extracellular acidification rates (ECARs) of iTregs (C) or CD4+CD25− Teffs (D) from WT or TKO mice after 12 weeks of HFD as measured by Seahorse (n = 4 per group). (E and F) Oxidative phosphorylation (OXPHOS) complexes I–V were detected by western blot analyses and quantified using Image J of iTregs (E) or CD4+CD25− Teffs (F) from WT or TKO mice after 12 weeks of HFD (lysates from n = 4 mice were pooled for each group). (G and H). Histograms represent MitoTracker staining of iTregs (G) or CD4+CD25− Teffs (H) from WT or TKO mice after 12 weeks of HFD. Bar graphs represent MitoTracker percentages in WT and TKO groups compared to unstained control group (n = 6 per group). Statistical differences are indicated as *p<0.05, **p<0.01, and *** p<0.001. Results are reported as mean ± SEM.

References

    1. Becker M, Levings MK, and Daniel C (2017). Adipose-tissue regulatory T cells: Critical players in adipose-immune crosstalk. Eur. J. Immunol 47, 1867–1874. - PubMed
    1. Beier UH, Angelin A, Akimova T, Wang L, Liu Y, Xiao H, Koike MA, Hancock SA, Bhatti TR, Han R, et al. (2015). Essential role of mitochondrial energy metabolism in Foxp3+ T-regulatory cell function and allograft survival. FASEB J. 29, 2315–2326. - PMC - PubMed
    1. Bertola A, Bonnafous S, Anty R, Patouraux S, Saint-Paul MC, Iannelli A, Gugenheim J, Barr J, Mato JM, Le Marchand-Brustel Y, et al. (2010). Hepatic expression patterns of inflammatory and immune response genes associated with obesity and NASH in morbidly obese patients. PLoS One 5, e13577. - PMC - PubMed
    1. Bluestone JA, Mackay CR, O’Shea JJ, and Stockinger B (2009). The functional plasticity of T cell subsets. Nat. Rev. Immunol 9, 811–816. - PMC - PubMed
    1. Brestoff JR, and Artis D (2015). Immune regulation of metabolic homeostasis in health and disease. Cell 161, 146–160. - PMC - PubMed

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