Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation
Samantha E. Adamson, Rachael Griffiths, Radim Moravec, Subramanian Senthivinayagam, Garren Montgomery, Wenshu Chen, Jenny Han, Poonam R. Sharma, Garrett R. Mullins, Stacey A. Gorski, Jonathan A. Cooper, Alexandra Kadl, Kyle Enfield, Thomas J. Braciale, Thurl E. Harris, Norbert Leitinger
Samantha E. Adamson, Rachael Griffiths, Radim Moravec, Subramanian Senthivinayagam, Garren Montgomery, Wenshu Chen, Jenny Han, Poonam R. Sharma, Garrett R. Mullins, Stacey A. Gorski, Jonathan A. Cooper, Alexandra Kadl, Kyle Enfield, Thomas J. Braciale, Thurl E. Harris, Norbert Leitinger
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Research Article Immunology

Disabled homolog 2 controls macrophage phenotypic polarization and adipose tissue inflammation

Abstract

Acute and chronic tissue injury results in the generation of a myriad of environmental cues that macrophages respond to by changing their phenotype and function. This phenotypic regulation is critical for controlling tissue inflammation and resolution. Here, we have identified the adaptor protein disabled homolog 2 (DAB2) as a regulator of phenotypic switching in macrophages. Dab2 expression was upregulated in M2 macrophages and suppressed in M1 macrophages isolated from both mice and humans, and genetic deletion of Dab2 predisposed macrophages to adopt a proinflammatory M1 phenotype. In mice with myeloid cell–specific deletion of Dab2 (Dab2fl/fl Lysm-Cre), treatment with sublethal doses of LPS resulted in increased proinflammatory gene expression and macrophage activation. Moreover, chronic high-fat feeding exacerbated adipose tissue inflammation, M1 polarization of adipose tissue macrophages, and the development of insulin resistance in DAB2-deficient animals compared with controls. Mutational analyses revealed that DAB2 interacts with TNF receptor–associated factor 6 (TRAF6) and attenuates IκB kinase β–dependent (IKKβ-dependent) phosphorylation of Ser536 in the transactivation domain of NF-κB p65. Together, these findings reveal that DAB2 is critical for controlling inflammatory signaling during phenotypic polarization of macrophages and suggest that manipulation of DAB2 expression and function may hold therapeutic potential for the treatment of acute and chronic inflammatory disorders.

Authors

Samantha E. Adamson, Rachael Griffiths, Radim Moravec, Subramanian Senthivinayagam, Garren Montgomery, Wenshu Chen, Jenny Han, Poonam R. Sharma, Garrett R. Mullins, Stacey A. Gorski, Jonathan A. Cooper, Alexandra Kadl, Kyle Enfield, Thomas J. Braciale, Thurl E. Harris, Norbert Leitinger

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Figure 6

DAB2 regulates NF-κB activity by suppressing phosphorylation of p65 (Ser536).

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DAB2 regulates NF-κB activity by suppressing phosphorylation of p65 (Ser...
(A) HEKTLR2 cells were cotransfected with an IL8 promoter–luciferase reporter construct and either pCGT (control) or pCGT–DAB2 p96 and then treated overnight with control media only (control) or LTA (1 μg/ml). (B) HEKTLR2/NF-κB–luc cells were transfected with either pCGT or pCGT-DAB2 p96 and then stimulated with LTA (1 μg/ml) overnight. Luciferase activity was measured by luminometry (BioTek Synergy HT). (A and B) *P < 0.001, by 2-way ANOVA with Sidak’s multiple comparisons test. (C) Immunoblot analysis of total p65 and p-p65 (Ser536) in pCGT and pCGT-DAB2–transfected HEKTLR2 cells that were treated with 1 μg/ml LTA for 0, 5, 15, 30, and 60 minutes. Actin served as a loading control. (D) BMDMs from Dab2fl/fl and Dab2fl/fl Lysm-Cre mice were treated with LTA for the indicated times, and protein lysates were immunoblotted using Abs to detect p-p38 (T180/Y182), total p38, p-p65 (Ser536), total p65, p-IKKβ (Ser177), and β-actin. (E) Dab2fl/fl and Dab2fl/fl Lysm-Cre mice were injected i.p. with 2 mg/kg LPS (E. coli 0111:B4). After 4 hours, peritoneal cells were isolated and stained with anti–F4/80 Alexa Fluor 647, anti–CD11b Alexa Fluor 488, and anti–p-p65 (Ser536) Abs. F4/80hiCD11bhi cells were gated, and the MFI of Ser536 p-p65–specific fluorescence was analyzed. Data represent the mean ± SEM; n = 7. *P < 0.05, by 2-tailed, unpaired Student’s t test. (F) NF-κB DNA–binding activity in nuclear extracts from Dab2fl/fl and Dab2fl/fl Lysm-Cre BMDMs treated with 100 ng/ml LPS for 1 hour was analyzed by TransAM EMSA (Active Motif). For competitive binding studies, functional (oligo) or nonfunctional (oligo-mut) oligonucleotides were used according to the manufacturer’s instructions. Data are expressed as NF-κB–binding activity at A450 to A650 nm (mean ± SD) of triplicate measurements. A, absorbance. *P < 0.0001 and #P < 0.02 compared with control and compared with LPS plus oligonucleotide, by 2-tailed, unpaired Student’s t test.