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, 38 (1), 303

Intestinal Dysbacteriosis-Induced IL-25 Promotes Development of HCC via Alternative Activation of Macrophages in Tumor Microenvironment

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Intestinal Dysbacteriosis-Induced IL-25 Promotes Development of HCC via Alternative Activation of Macrophages in Tumor Microenvironment

Qiao Li et al. J Exp Clin Cancer Res.

Abstract

Background: Gut microbiota and the tumor microenvironment are thought to be critical factors that modulate the processes of liver diseases, including hepatocellular carcinoma (HCC). Interleukin-25 (IL-25) promotes type 2 immunity via alternative activation of macrophages, and is closely associated with inflammation-related diseases, even malignancies. However, it is not clear which role IL-25 plays in the development of HCC, and whether gut microbiota are involved.

Methods: IL-25 was detected by ELISA, Western blotting (WB), and immunohistochemistry. Chemokines were measured by RT-qPCR and WB. After co-culture with IL-25-stimulated macrophages, the cell growth, migration, invasion and EMT marker of HCC cell lines (MHCC97L and HepG2) were evaluated by Brdu proliferation, Transwell assays and WB. An antibody neutralization assay of chemokine CXCL10 was performed to confirm its role in HCC development. Furthermore, the effects of IL-25 in HCC were investigated in vivo. Dysbiosis of gut microflora was induced by antibiotics (vancomycin, cefoperazone or combination of ampicillin, neomycin, metronidazole, and vancomycin). We used feces suspension to treat colonic epithelial NCM460 cells, and detected IL-25 and tuft cell marker DCLK1 using WB and immunofluorescence staining.

Results: We found that the level of IL-25 was significantly elevated in HCC patients, and was negatively correlated with survival rate after hepatectomy. However, IL-25 did not directly promote the development of HCC cells. Then, we observed the significant positive correlation between IL-25 level and M2 percentage (CD206/CD68) in HCC tumors. In vitro and in vivo, IL-25 induced alternative activation of macrophages promoted HCC cell migration, invasion and tumorigenesis, increased the expression of vimentin, Snail and phospho-ERK, and decreased the expression of E-cadherin in HCC cells. After IL-25 treatment, chemokine CXCL10 was increased in macrophages. Neutralizing CXCL10 in macrophage-conditioned medium reversed the IL-25-mediated effect on HCC cells. Vancomycin-induced dysbiosis promoted the growth of orthotopic HCC homograft. Surprisedly, we found the hyperplasia of colonic epithelial tuft cells, from which more IL-25 was secreted .

Conclusions: IL-25 promotes the progression of HCC through inducing alternative activation and CXCL10 secretion of macrophages in tumor microenvironment, and IL-25 secretion may partly result from hyperplastic epithelial tuft cells in colon, induced by gut microbiota dysbiosis.

Keywords: Chemokine; Gut microbiota; Hepatocellular carcinoma; Interleukin-25; Macrophages; Tumor microenvironment.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Overexpression of IL-25 is found in HCC patients, and predicts the poor prognosis. a Serum levels of IL-25 were detected by ELISA in hepatic hemangioma patients (n = 5) and HCC patients (n = 10). b Concentrations of IL-25 were detected by ELISA in tissue homogenates of normal liver tissues (n = 5) and HCC tumor tissues (n = 10). c Protein levels of IL-25 were detected by Western blotting in normal liver tissues (n = 4) and HCC tumor tissues (n = 4). d and e Immunohistochemistry (IHC) staining of IL-25 was performed in a tissue microarray consisted of 98 HCC tumor tissues and 55 normal liver tissues. d IL-25 representative IHC images (left) with statics (right) of the corresponding levels are shown. Bar, 20 μm (up), 100 μm (down). Overall survival (e) and disease free survival (f) curves of 98 HCC patients in correlation with intra-tumor IL-25 scores. The third quartile of the IL-25 scores was used as a cut-off value. The patients with HCC were divided into 2 groups according to the intra-tumor IL-25 score: low group (n = 70), high group (n = 28). The clinical characteristics of these two group are summarized in Additional file 1: Table S2. *p < 0.05, ***p < 0.001
Fig. 2
Fig. 2
IL-25 activates alternative macrophages (M2), which promote HCC cell migration and invasion. a IL-25 was used to directly treated HCC cells in vitro, the relative value of CCK-8, Annexin-V, Brdu, Transwell experiment. (Please see results in Additional file 1: Figure S1A-D). b Immunofluorescent staining was performed in normal liver tissues (n = 55) and HCC tumor tissues (n = 98). Representative immunofluorescence images. Bar, 50 μm. c Immunohistochemistry staining was performed in a HCC tissue microarray (n = 98). Correlation between M2 macrophage (CD206/CD68) percentage and IL-25 scores. CD206 is an M2 macrophage marker, and CD68 is macrophage marker. d and e Macrophages (derived from THP-1) were treated with IL-25 in a time- and concentration-dependent manner. Tumor necrosis factor-α (TNF-α) and CD206 were examined by Western blotting. f and i HCC cells co-cultured with M0 or M2 macrophages. f Cell growth was determined with the Brdu kit. Statistical data were shown at the right. Bar, 100 μm. HCC cells migration (g) and invasion (h) were determined by Transwell assay. Statistical data were shown at the right. Bar, 100 μm. i Mesenchymal maker vimentin, EMT regulator Snail, epithelial marker E-cadherin, extracellular signal-regulated kinase (ERK), and p-ERK were detected by Western blotting. ***p < 0.001, ns, no significance
Fig. 3
Fig. 3
IL-25-induced M2 macrophages promote the EMT process in HCC cells via CXCL10. a-d Macrophages (derived from THP-1) were treated with IL-25 in a time- and concentration-dependent manner. a and b CXCL10 gene expression was quantified by RT-qPCR. c and d CXCL10 protein level was determined by Western blotting. e-g HCC cells were co-cultured with M0 or M2 macrophages. Then, anti-CXCL10 antibody was added to the M2 macrophage culture medium to neutralize CXCL10 protein. Migration (e) and invasion (f) of HCC cells were determined by Transwell assay. Statistical data were shown at the right. Bar, 100 μm. g Mesenchymal maker vimentin, EMT regulator Snail, epithelial marker E-cadherin, extracellular signal-regulated kinase (ERK), and p-ERK were detected by Western blotting. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance
Fig. 4
Fig. 4
IL-25-induced M2 macrophages promote tumorigenesis and EMT of HCC cells in vivo. a Images of tumors from each group. b Tumor weight at the time of sacrifice. c Tumor necrosis factor-α (TNF-α) and CD206 in the tumor tissue of each group were detected by Western blotting. Statistical data were shown at the right. d Mesenchymal maker vimentin, EMT regulator Snail, epithelial marker E-cadherin, extracellular signal-regulated kinase (ERK), and p-ERK were detected in the tumor tissue of each group by Western blotting. Statistical data were shown at the right. e Chemokine CXCL10 was detected in the tumor tissue of each group by Western blotting. Statistical data were shown at the right. *p < 0.05, **p < 0.01, ns, no significance
Fig. 5
Fig. 5
Gut bacterial dysbiosis promotes hyperplasia of tuft cells and secretion of IL-25. a-e An orthotopic C57BL/6 mice hepatic tumor model with gut microflora dysbiosis was prepared as described in the methods. a Images of tumors from each group. b Tumor weight at the time of sacrifice. c Serum level of IL-25 was detected by ELISA in each group. d Concentrations of IL-25 in small intestine and colon tissue homogenates were detected by ELISA. e Tuft cell marker DCLK1 representative IHC images. Statistical data were shown at the right. Bar, 20 μm. f-h 16S rRNA sequencing and analysis of feces gut microbiota of mice. N, normal control group. A, combination antibiotics group (ANMV). C, cefoperazone group. V, vancomycin group. f PCoA score based on weighted unifrac metrics was different in each group. g Observed bacterial species’ richness in feces samples from each group. p < 0.05 by Wilcoxon rank-sum test. h Hierarchical clustering of each group using Bray-Curtis dissimilarity indices at the phylum level by the weighted unifrac distances. i and j Feces suspensions with bacteria from the above groups were used to treat colonic epithelial NCM460 cells. i Western blotting was performed to determine DCLK1 and IL-25 levels. j Immunofluorescence staining was used to detect DCLK1 (red) and IL-25 (green) in slides embedded with NCM460 cells. Representative immunofluorescence images. Bar, 50 μm. k Feces suspensions without bacteria from the above groups were used to treat colonic epithelial NCM460 cells. Western blotting was performed to detect DCLK1 and IL-25 levels. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance
Fig. 6
Fig. 6
Gut bacterial dysbiosis results in colonic epithelial tuft cell hyperplasia and increased secretion of IL-25, which enters the liver via portal vein. IL-25 derived from the gut promotes the alternative activation of macrophages and fosters the tumorigenesis and migration of HCC cells via chemokine CXCL10

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