TET3-overexpressing macrophages promote endometriosis
Haining Lv, Beibei Liu, Yangyang Dai, Feng Li, Stefania Bellone, Yuping Zhou, Ramanaiah Mamillapalli, Dejian Zhao, Muthukumaran Venkatachalapathy, Yali Hu, Gordon G. Carmichael, Da Li, Hugh S. Taylor, Yingqun Huang
Haining Lv, Beibei Liu, Yangyang Dai, Feng Li, Stefania Bellone, Yuping Zhou, Ramanaiah Mamillapalli, Dejian Zhao, Muthukumaran Venkatachalapathy, Yali Hu, Gordon G. Carmichael, Da Li, Hugh S. Taylor, Yingqun Huang
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Research Article Inflammation Reproductive biology

TET3-overexpressing macrophages promote endometriosis

Abstract

Endometriosis is a debilitating, chronic inflammatory disease affecting approximately 10% of reproductive-age women worldwide with no cure. While macrophages have been intrinsically linked to the pathophysiology of endometriosis, targeting them therapeutically has been extremely challenging due to their high heterogeneity and because these disease-associated macrophages (DAMs) can be either pathogenic or protective. Here, we report identification of pathogenic macrophages characterized by TET3 overexpression in human endometriosis lesions. We show that factors from the disease microenvironment upregulated TET3 expression, transforming macrophages into pathogenic DAMs. TET3 overexpression stimulated proinflammatory cytokine production via a feedback mechanism involving inhibition of let-7 miRNA expression. Remarkably, these cells relied on TET3 overexpression for survival and hence were vulnerable to TET3 knockdown. We demonstrated that Bobcat339, a synthetic cytosine derivative, triggered TET3 degradation in both human and mouse macrophages. This degradation was dependent on a von Hippel-Lindau (VHL) E3 ubiquitin ligase whose expression was also upregulated in TET3-overexpressing macrophages. Furthermore, depleting TET3-overexpressing macrophages either through myeloid-specific Tet3 ablation or using Bobcat339 strongly inhibited endometriosis progression in mice. Our results defined TET3-overexpressing macrophages as key pathogenic contributors to and attractive therapeutic targets for endometriosis. Our findings may also be applicable to other chronic inflammatory diseases where DAMs have important roles.

Authors

Haining Lv, Beibei Liu, Yangyang Dai, Feng Li, Stefania Bellone, Yuping Zhou, Ramanaiah Mamillapalli, Dejian Zhao, Muthukumaran Venkatachalapathy, Yali Hu, Gordon G. Carmichael, Da Li, Hugh S. Taylor, Yingqun Huang

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Figure 4

Bc induces apoptosis of TET3 OE macrophages.

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Bc induces apoptosis of TET3 OE macrophages. (A) Human MDMs were incubat...
(A) Human MDMs were incubated with Veh or Bc at a final concentration of 10 μM for 2 hours, followed by time-course analysis of TET3 in the presence of CHX at a final concentration of 50 μg/mL. Cells were harvested at 0, 20, 40, and 60 minutes after addition of CHX. Human MDMs in CM-Endo (B) and RAW 264.7 cells (C) were incubated with Veh or Bc at a final concentration of 10 μM for 24 hours. Proteins were extracted and analyzed. (D) Immunoblots for TET3 and VHL in MDMs pretreated with or without VHLprotac at the concentration of 5 μM for 18 hours, followed by exposure to Bc at 10 μM for 8 hours. (E) PMs from WT mice primed with 30 ng/mL of TGF-β1 were conditioned by VHLprotac 5 μM for 18 hours, followed by exposure to Bc at 10 μM for 8 hours and Western blot analysis. (F) co-IP of Flag-TET3 and endogenous VHL in H1299 cells transfected with Ad-TET3, with or without the presence of Bc at 50 μM for 2 hours. MDMs (G and H) (primed with 10 ng/mL of TGF-β1) and RAW 264.7 cells (I and J) were incubated with Veh plus GFP-expressing adenovirus (Veh+Ad), Bc at 10 μM plus Ad (Bc+Ad), or Bc at 10 μM plus TET3-expressing adenovirus (Bc+Ad-TET3) for 48 hours, followed by immunoblotting and TUNEL assays. Representative immunoblots, photomicrographs and corresponding statistical analysis are shown. For TUNEL assay, n = 3 randomly selected areas per group. All data are represented as mean ± SEM. ***P < 0.001, 1-way ANOVA with Tukey’s post test (H and J). Scale bars: 40 μm. The dashed dividing lines (A and F) indicate splicing of noncontiguous lanes from the same blots.