Effect Of Transmembrane And Secretory TNF-αon Malignant Transformation From Nonresolving Inflammation

In the progression of malignant transformation from nonresolving inflammation, the pro-inflammatory factors may be involved in tumor initiation, but the anti-inflammatory factors can promote tumor progression through the inhibition of antitumor immunity. Soluble tumor necrosis factor-a (sTNF-a), a pro-inflammatory cytokine, has been shown to play an important role in tumorigenesis and tumor development via promotion of inflammation. However, the action of transmembrane TNF-a (tmTNF-a) is still obscure in this pathological process. In this study, we used AOM/DSS to induce colitis-associated carcinogenesis in mice and observed the dynamic variation of both forms of endogenous TNF-α in different stages (acute inflammation, chronic inflammation and tumor stage) of this pathological process. We also analyzed the relevance between both forms of endogenous TNF-α and temporal and spatial variation of pro-inflammatory and anti-inflammatory cells and cytokines. We observed if tmTNF-α expression window would be advanced to the acute inflammation stage by treatment of mice with tmTNF-α antibody, which may relieve malignant transformation. This study not only elucidates the role of tmTNF-α in this pathological process, but also provides a new strategy and a new clue for the early diagnosis and blockage of this pathological process. The main results are as follows:I. The relevance of endogenous transmembrane and secretory TNF-α to pro-and anti-inflammatory cells and molecules in the process of malignant transformation from nonresolving inflammation1. The model of chronic colitis-associated colon cancer:(1) Clinical changes in chronic colitis-associated colon cancer:C57BL/6mice intraperitoneally (ip) administered with AOM7days in advance were treated with three cycles of2.5%DSS in drinking water for5days and regular water for14days. After each cycle of giving2.5%DSS drinking, mice showed weight loss, colon shortening, fecal occult blood, and even rectal bleeding, but these symptoms could be ameliorated after two weeks of normal water.(2) Tumors induced by chronic colitis:The third cycle is the malignant stage, and tumors are very frequent in the distal part of the colon. The average amount of each tumor-bearing mice is about10, and the average diameter of tumor is about4-5mm.(3) The colon pathological changes in chronic colitis-associated colon cancer:The first cycle is acute inflammation stage, and HE staining showed that the intestinal epithelium is missing and a lot of inflammatory cells infiltrate into the colon lamina propria in this stage; but the intestinal structure is nearly normal after2weeks. The second cycle is chronic inflammation stage; in this stage, inflammatory cells can infiltrate into the submucosa and the hyperplasia of intestinal glands can be observed after2weeks. The third cycle develops early adenomas, and the glandular cells are arranged irregularly; then malignant cells become increased in later and enter the tumor stage.2. Changes of pro-and anti-inflammation cells in chronic colitis-associated colon cancer:(1) The number and phenotype changes of pro-and anti-inflammation cells in chronic colitis-associated colon cancer:Flow cytometry and immunohistochemistry results showed that the macrophage number of spleen and mesenteric lymph nodes reached a peak in the acute inflammation recovery stage and malignant stage, but the number reduced in the tumor stage. Macrophages in colonic tissues including M1and M2macrophages were significantly elevated in malignant transformation of chronic inflammation. Spleen Th17cells were increased in varying degrees throughout the pathological process, but they reached a peak in the recovery phase of acute inflammation. While Th17cells in mesenteric lymph nodes and colon tissues reached a peak in the tumor stage.(2) Treg cells in chronic colitis-associated colon cancer:Flow cytometry and immunohistochemistry results showed that spleen Treg cells has risen to a high level in acute inflammation, but significantly decreased in the tumor stage. Treg cells in mesenteric lymph nodes increased only when it is in the DSS-induced damage, but decreased in the interim period. Treg cells in colonic tissues were significantly higher in the malignant transformation from nonresolving inflammation, and can infiltrate into the submucosa.3. Changes of pro-and anti-inflammatory molecules in chronic colitis-associated colon cancer:(1) Th1-and Th2-type cytokines:ELISA results showed that IFN-γ in colon tissue culture supernatants and colon tissue homogenates was increased in inflammation damage induced by DSS and reduced in the intermittent period. The IL-4level is very low and its dynamic changes are opposite to IFN-γ. It suggests that response in this model is mainly Thl-mediated inflammation.(2) Pro-and anti-inflammatory cytokines in chronic colitis-associated colon cancer: ELISA and Griess results showed that pro-inflammatory cytokines including IL-1β, IL-6and NO in colon tissue culture supernatants and colon tissue homogenates mainly were elevated during the DSS-induced inflammation damage, but significantly decreased in the intermittent. Conversely, anti-inflammatory molecules IL-10and TGF-β in colon tissue homogenates were low during the DSS-induced inflammation damage and increased in the intermittent. But the trends of IL-10and TGF-β in colon tissue culture supernatants were contrary with those in colon tissue homogenates. In addition, we found that the magnitude increase of anti-inflammatory molecules was higher than pro-inflammatory molecules in the tumor stage. This suggests an important role of anti-inflammatory cytokines in tumor development.4. Changes of both forms of TNF-α in chronic colitis-associated colon cancer:(1) TNF-α in serum and colon tissue:ELISA results showed that serum sTNF-α was elevated slightly in acute inflammation stage, reached a peak in recovery, then declined and peaked again in tumor stage. But total TNF-α in tissue homogenates increased in acute inflammation stage, then decreased and increased again in tumor stage.(2) Transmembrane and secretory TNF-α ratio in colon tissue:ELISA results confirmed that sTNF-α in colon tissue culture supernatants increased significantly in acute and chronic inflammation, so the ratio of tmTNF-α/sTNF-α was significantly lower. As the expression of tmTNF-α in colon tissue membrane proteins was increased in the malignant transformation from chronic inflammation, the proportion of the ratio also increased.(3) tmTNF-α-expressing cell origin in colon tissue:Immunohistochemistry results showed that it is mainly infiltrating cells expressing tmTNF-α in acute and chronic inflammation stage, but tmTNF-α was mainly expressed by adenomatous hyperplasia epithelial cells in the malignant transformation stage. Isolated colonic epithelial cells showed that tmTNF-α expression was up-regulated during malignant transformation from nonresolving inflammation. Therefore tmTNF-α may be of warning significance for malignant transformation.(4) Correlation between both forms of TNF-α and pro-and anti-inflammatory cytokines:Colon tissue sTNF-α levels were positively correlated to the levels of pro-inflammatory cytokines as IL-1β, IL-6and NO in colon tissue homogenates, but negatively correlated to the levels of anti-inflammatory cytokines IL-10and TGF-β. While colon tissue tmTNF-α levels were negatively correlated to the levels of pro-inflammatory cytokines IL-1β, IL-6and NO in colon tissue homogenates, and positively correlated to anti-inflammatory cytokines IL-10.(5) A correlation between tmTNF-α expression and Treg accumulation:The expression of tmTNF-α was positively related to the accumulation of Foxp3+Treg in colon tissues.Ⅱ. The effect of tmTNF-α antibody on the malignant transformation from nonresolving inflammation1. tmTNF-α antibody effectively blocks tmTNF processing in vivo:Treatment of mice with tmTNF-a antibody on the day of the first cycle DSS withdrawal twice a week and600μg per mice. It confirmed that tmTNF-α antibody significantly increased tmTNF-a expression and reduced sTNF-α secretion in colon tissue lesions.2. tmTNF-α antibody relieves malignant transformation:Treatment of mice with tmTNF-α antibody at the acute inflammation stage could reduce the number and volume of colitis-associated tumors and splenomegaly of the model mice, but could not improve some clinical symptoms such as body weight loss or fecal bleeding induced by DSS.3. The effect of tmTNF-a antibody on pro-and anti-inflammatory cytokine production in colon tissue lesions:Treatment of mice with tmTNF-α antibody could not affect the content of IFN-Y and IL-4in homogenates and culture supernatants of colon tissue lesions, but could inhibit the production of IL-1β, IL-6and NO. It could also significantly increase the IL-10levels in homogenates and culture supernatants of colon tissue lesions, but had no influence on producing of TGF-β.4. The impact of tmTNF-α antibody on the number of macrophages and Th17cells in colon tissue lesions:Immunohistochemistry results confirmed that treatment of mice with tmTNF-α antibody could inhibit infiltration of macrophages, mainly M1type macrophages, but it has no effect to on M2type macrophages’infiltration. So it has no significant effect on IL-17positive cells.In summary, the mucosal and systematic immunity are all involved in the progression of malignant transformation from nonresolving inflammation. We found that sTNF-a was mainly increased at acute inflammation stage and had a correlation with pro-inflammatory cells and molecules. While tmTNF-α began to become heightened at the stage of nonresolving inflammation and had a correlation with anti-inflammatory cells and molecules. The data suggested that sTNF-α promotes tumorigenesis by promoting inflammation, but tmTNF-α promotes tumor development by suppressing immune. TmTNF-α expression window could be advanced to the inflammation stage by treatment with tmTNF-α antibody, and it may relieve malignant transformation. This study not only clarifies a novel molecular mechanism involved in malignant transformation from nonresolving inflammation, and also provides a new clue for blockage of this pathological process using a new targeting antibody.