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  • br We next performed microarray analysis of


    We next performed microarray analysis of mouse endometrium under estrogen and high-fat diet intervention and found a gene set as-sociated with ubiquitin enzymes was dysregulated (Fig. 2C). CD163+ macrophage CM intervention further confirmed the same change in ubiquitin enzymes in HEC-1A Haloperidol (Supplementary Fig. S2). Among the ubiquitin-editing enzymes identified, A20 was markedly elevated in the CD163+ macrophage CM intervention test (Fig. 2D). As ubiquitination modification is an important post-translational mechanism for ERα protein modification [16], we thereby evaluated the potential re-lationship among A20, ERα expression, and the number of infiltrating CD163+ macrophages in human endometrial tissues (Fig. 2E). IHC staining showed small numbers of CD163+ macrophages in the stroma of normal proliferating endometrium. The number of CD163+ macro-phages increased gradually when endometrial lesions developed from hyperplasia to EC (Fig. 2F). Since the stroma gradually decreased in the lesion tissues from G1 to G3, the amount of infiltrating CD163+ mac-rophages also consistently decreased in the same proportion in IHC staining. Consistent with CD163+ macrophage infiltration, A20 and ERα were both highly expressed and positively correlated in hyper-plasia, EAH, and EEC G1 (Fig. 2G), but decreased gradually when the lesions progressed from G1 to G3. We further analyzed A20 protein levels in ERα-positive or ERα-weak positive EC cell lines (Ishikawa, ECC1, RL95-2, and HEC-1A) and ERα-negative EC cell lines (KLE and 
    SPEC2) (Fig. 2H). ERα and A20 protein expression changed by a similar degree in EC cell lines. These findings suggested that A20 and ERα protein were positively correlated in the microenvironment of CD163+ macrophages.
    3.3. A20 and ERα are upregulated by CD163+ macrophages via cytokines
    To explore the potential mechanisms involved in CD163+ macro-phage-mediated upregulation of ERα protein, CD163+ macrophages and HEC-1A co-culture medium were collected to assess cytokine changes by cytokine array (Fig. 3A). Cytokines such as GM-CSF, IL1α, IL6, IL10, IL17A, MCP2, and TNFα, increased remarkably in the su-pernatant of co-culture systems compared with that from CD163+ macrophages or HEC-1A alone (Fig. 3B). We then asked whether CD163+ macrophages upregulated A20 and ERα in a paracrine manner through these cytokines. Interestingly, IL-1α, IL17A, and TNFα in-creased A20 and ERα expression in EC cells (Fig. 3C). We next selected IL17A for further investigation. IL17A increased A20 and ERα expres-sion in a dose- and time-dependent manner, and neutralization of IL17A with its antagonistic antibody blocked IL17A-induced A20 and ERα expression (Supplementary Fig. S3).
    As IL1α, IL17A, and TNFα regulate the NF-κB pathway, NF-κB in-hibitor PDTC was used to test whether this pathway is involved in the upregulation of A20 and ERα expression. Data showed that PDTC blocked cytokine-induced expression of A20 and ERα (Fig. 3D). IL1α and TNFα upregulated A20 transcripts without affecting ERα tran-scripts, while IL17A upregulated ERα transcripts very slightly (Fig. 3E). These findings suggested that IL1α and IL17A might upregulate A20
    Fig. 2. A20 and ERα are positively correlated with the amount of CD163+ macrophages in endometrial lesions. A. CD163+ macrophage culture medium (CM) upregulated ERα protein expression but slightly upregulated ESR1 mRNA levels in EC cells. Cells were cultured in normal medium (NM), THP1 CM, and CD163+ macrophage CM for 16 h and 36 h with the most significant effect for RT-PCR and western blotting. The fold change of ERα relative to control group was calculated from densitometry. B. CD163+ macrophage CM did not have significant transactivation effect on the ESR1 promotor in EC cells. Cells were transfected with pGL3-ESR1 promoter plasmids and pGL4.74 [hRluc/TK] (internal control) for 18 h and then cultured in normal medium (NM), THP1 CM, and CD163+ macrophage CM for 18 h. C. Ubiquitin enzymes in mouse endometrium regulated by prolonged estrogen and high-fat diet intervention. The samples of endometrium were collected for microarray analysis. The mRNA levels of TNFAIP3 were increased in the intervention group (black arrow). D. CD163+ macrophage CM upre-gulated mRNA and protein levels of A20 in EC cells. Cells were cultured in NM, THP1 CM, and CD163+ macrophage CM for 16 h and 36 h for RT-PCR and western blotting analysis. E. IHC staining for CD163, A20, and ERα expression in endometrial hyperplasic lesions. Original magnification 200 × . Scale bars, 100 μm. F. The number of CD163+ macrophages increased gradually from normal endometrium in the proliferative phase to EC. G. The staining intensity of A20 and ERα is scored by semi-quantitative optical analysis. Correlation of A20 and ERα IHC scores was analyzed by Spearman's correlation. H. ERα and A20 protein expression changed to a similar degree in EC cell lines. Baseline A20 and ERα protein expression in different EC cell lines was analyzed by western blotting. PP, proliferative phase; H, hyperplasia; EAH, endometrial atypical hyperplasia; G1, endometrioid adenocarcinoma grade 1; G2, endometrioid adenocarcinoma grade 2; G3, endometrioid adenocarcinoma grade 3. *P < 0.05, **P < 0.01, ***P < 0.001.