The Mechanistic Action Of Complement On Colitis-associated Colorectal Cancer Development

Complement system plays a very important role in maintaining stable micro-environment and host defense against infection. The complement components exist in the form of inactive enzymes precursor under physiological conditions untill being activated to mount cascade reaction by some factors. It is well established that complement system is involved in the pathogenesis of inflammation and tumor. Different from the positive effect on inflammation, complement system plays a paradoxical complicated role in tumor:some experimental results confirmed that complement inhibited tumor growth, while the others showed that complement system promoted tumor development.Ulcerative colitis (UC) is an chronic inflammatory bowel disease with unknown etiology, long duration period and lacking effective treatment. The most serious complication of UC is Colitis-associated colorectal cancer (CAC) and the incidence of CAC is related to the extension of disease. Approximately 15% of the patients died of cancer.Complement system is involved in inflammatory bowel disease and colorectal ;ancer, but few reports are to investigate complement role in the transformation of colitis into colorectal cancer. Thus, it is worthy to explore the role of complement system in CAC development.Objective To investigate the effect and underlying mechanism of complement on CAC development and provide rationale for targeting complement to treat CAC in clinic.Method 1. Using azoxymethane (AOM) and dextran sulfate sodium (DSS) to establish a mice model of CAC:AOM is injected intraperitoneally at a dose of 10mg kg; 5 days later 1.5% DSS solution was administrated orally for 7 days, then regular water is changed for 14 days.Thereafter, two more DSS treatment cycles were needed. Mice model of acute colitis is established by 3% DSS solution for 5 days.2. Complement system activation parameters were detected on day 0,20, 40,60 and 100 respectively. Intestinal mRNA expression levels of C3, C5, C3aR and C5aR was detected by quantitative polymerase chain reaction (qPCR). C3a and C5a levels in colons were examined by enzyme-linked immune sorbent assay (ELISA).C3 expression in lesions was also detected by immunohistochemistry;3. To observe tumor burden in complement-knockout mice (C3, C5, C5aR-knockout) and wild-type mice subjected to CAC induction;4. To explore the mechanisms by which complement activation promotes CAC development. Intestinal immune cell infiltration was detected by immunohistochemistry and flow cytometry respectively. Intestinal permeability and bacterial translocation was examined in model of acute colitis. Cytokines levels in colons were detected by QPCR and ELISA. Immune cells in the lamina porpria of colons were sorted by fluorescence activated cell sorting(FACS) and cultured with appropriate factors ex vivo. Intestinal epithelial proliferation/apoptosis and involving signaling pathways were detected by immunohistochemical and westen blotting.Results 1. CAC and acute colitis mice model were established successfully as reflected by several parameters such as diarrhea, blood stool and weight loss. Histopathological examination showed visible intestinal congestion, edema, epithelial visible erosion, ulceration of lesions as well as hyperplasia and malignant transformation in CAC model.2. Compared with the control group, complement expression (C3, C5, C3aR, C5aR) and complement activation products(C3a, C5a) levels in colons increased significantly in CAC model. C5a expression increased on day 20 and 40 then declined, because C5a bound to C5aR leading to internalization. These results showed that during the progression from colitis to CAC complement system was sustainedly activated.3. Compared with wild-type mice, tumor number in complement-knockout mice(C3, C5 or C5aR) were much lower, suggesting that during the progression from colitis to CAC complement system is indispensible for the development of CAC.4. Compared with wild-type mice, intestinal interleukin-1β (IL-1β), interleukin-17A(IL-17A) and interleukin-22 (IL-22) levels were significantly reduced in complement-deficient CAC-bearing mice, while no alterations of several putatively CAC-promoting factors(e.g. IL-6, tumor necrosis factor-a (TNF-a), interferon-γ (IFNy)) were observed. These results suggest that complement may regulate the IL-1β, IL-17A and IL-22 expression thereby promote tumorigenesis.To determine whether decreased neutrophil infiltration in complement-deficient mice was due to impaired epithelial barrier, acute colitis model was used and the results showed that intestinal permeability and bacteria translocation in complement-deficient mice was comparable to complement-suficient littermates suggesting that decreased neutrophil infiltration might be not attributed to alteration in intestinal epithelial barrier. Futhermore, we found C5aR expression in neutrophil, indicating a direct effect of C5a on neutroplil infiltration. Neutrophil was identified as main producer for IL-1β in lesions. Neutrophil infiltration and its expression of IL-1β were significantly decreased in complement-knockout mice. IL-17A was expressed predominantly by CD11b+ myeloid cells and IL-17A expression was dramatically decreased in these cells in complement-knockout mice. IL-1β was able to elicit IL-17A expression in myeloid cells. This effect was abrogated by addition of IL-1 receptor antagonist (IL-1Ra). Similar results in vivo were obtained. Conversely, injection of recombinant IL-1β led to increased IL-17A expression in intestinal myeloid cells suggesting that IL-17A expression was regulated by IL-1β. Neutrophils isolated from the lesions of CAC-beraing mice enable increased expression of IL-17A in myeloid cells while neutrophils from the complement-deficient mice did not. Overall, these findings suggested that during the progression from colitis to CAC, neutrophil-derived IL-1β regulate the expression of IL-17A in myeloid cells, confirming the existence of IL-1β/IL-17A axis in the intestine.IL-17A had capacity to promote the proliferation of CT26 cell line in a dose-dependent manner. Futhermore, phosphorylation levels of signal transducer and activator of transcription 3 (stat3) and nuclear factor κB (NF-κB) p65 were significantly reduced in complement-deficient mice, accompanied by increased apoptosis and decreased proliferation in intestinal epithelial.Conclusion Complement activation promotes CAC development. The schematic model is following:complement-activated products such as C5a caused neutrophil infiltration and production of large amounts of IL-1β in the lesions. IL-1β drived expression of IL-17A in intestinal myeloid cells. Futhermore, IL-17A activated stat3 and NF-κB signaling and ultimately led to the occurrence and development of colorectol cancer. Overall, complement activation promotes CAC development through activating intestinal IL-1β/IL-17A axis.