The Impact Of Myeloid Cells Myd88 Signaling In Colon Cancer

Objective:Because of the change of life style and diet habit,the morbidity and mortality of colorectal cancers in china increased these years,the morbidity of colorectal cancers in china is only lower than gastric cancers,lung cancers and esophagus cancers,and be the forth one.In the world the morbidity and mortality of colorectal cancers are among the top three of common fatal cancers.It has been long recognized that myeloid cells play an essential role in maintaining stable intestinal immune system and modulating colitis and CAC.Myeloid cells TGF-?signaling facilitates the development of CAC through the production of more proinflammatory agents and recruitment of more macrophages.COX-2 and av integrin deletion in myeloid cells exacerbates colitis in mice.Myeloid cells I??? deletion decreases tumor dimensions in mice treated with AOM/DSS,so myeloid cells modulate colitis and CAC through different signal pathways.It has been long recognized that MyD88 play an essential role in maintaining stable intestinal immune system and modulating colitis and CAC.MyD88 "whole-body"knockout system can certify MyD88 promotes cancer development,For example,Apc min/+MyD88-/-mice develop fewer colon tumors after many injections of AOM;AOM treatment only can cause IL10-/-MyD88-/-mice to develop fewer colon tumors as well.However,MyD88 signaling could limit the occurrence of tumors in more serious inflammatory environments,such as AOM/DSS-induced colorectal cancers.MyD88 signaling has also been shown to protect mice from developing colon cancer through promoting DNA repair,enhancing epithelial cell proliferation,and decreasing epithelial cell apoptosis.Considering that many results were based on MyD88 "whole-body"knockout system,therefore,the role of myeloid MyD88 in tumorigenesis is still largely unclear.MyD88 plays different roles under different circumstances in different cells.For non-immune system,it has been reported that epithelial-specific MyD88 deficiency results in age-related spontaneous chronic small intestine inflammation.On the other hand,for im-mune system,it has been suggested that T cell intrinsic MyD88 deficiency protects mice from CD4+T cell-induced colitis,whereas B cell-specific MyD88 signaling deficiency promotes DSS-induced colitis.Furthermore,in a mouse line with MyD88 expression restricted to myeloid lineages,it has been demonstrated that MyD88-dependent signaling can protect the intestine from DSS-induced tissue damage via promoting epithelial cell proliferation.However,far less is known about the effects of MyD88 signaling specific deficiency in myeloid cells in an AOM/DSS-induced CAC mouse model.We prepared CRC model in LysmCre MyD88FL/FL mice by AOM/DSS treatment,analyzed the sense of myeloid cells Myd88 signaling,and clarified the role of myeloid cells Myd88 signaling in colorectal cancer developing.The results will broaden the view and the role of myeloid cells Myd88 signaling.Method:Mice:LysmCre MyD88FL/FLmice and WT mice.Preparation of CAC mouse model:We use AOM/DSS to induce CAC in LysmCre MyD88FL/FLmice and WT mice(treated by AOM injection and then three cycles of DSS in water),detecting tumors(tumor numbers,tumor load,tumor pathology)of colon cancer by gross and by histologic analysis.HE dyeing:We detectd intestinal inflammation in early stage and tumor in advanced cancer of LysmCre MyD88FL/FL mice and WT mice by pathological examination.We detected proinflammation,mitogenic,angiogenic and pro-tumorigenic cytokines of large intestine in LysmCre MyD88FL/FL mice and WT mice by RT-PCR and we detect their weight loss,diarrhea scores,bleeding scores and DAI.FACS analyze:We analyzed immune cells of LysmCre MyD88FL/FL mice and WT mice,such as CD45+ cells,neutrophils,monocytes,macrophages,eosinophils,dendritic cells,CD4+T cells,CD8+T cells,Treg in colon lamina propria and MLN;We analyzed neutrophils in blood,colon lamina propria,BM;We analyzed hematopoietic stem cells and progenitors in BM.And we sorted neutrophils in colon lamina propria to detect cytokines.We analyzed Treg cells in colon lamina propria and SP of Lysmcre MyD88FL/FL mice and WT mice,we sorted CD8+T cells to detect cytokines secreted by them.After AOM/DSS treatment,we detected large intestinal epithelial cell proliferation by Ki67,detect large intestinal epithelial cell apoptosis by Tunel staining in LysmCre MyD88FL/FL mice and WT mice.Immunohistological staining and Western blot analysis:mucosal expression of COX-2,phospho-STAT3,?-catenin and cyclinDl in LysmCre MyD88FL/FL mice and WT mice were examined by western blot analysis and immunohistological staining after AOM/DSS treatment.qRT-PCR was performed to detect and analyze DNA repair gene(such as Parp1,mlhl,ATM and ATR)expression in large intestine of LysmCre MyD88FL/FL mice and WT mice.Exon 3 of ?-catenin mutations were also analyzed.Result:LysmCreMyD88FL/FL mice have higher tumor incidence than WT mice after AOM/DSS administration.Histologic analysis showed that LysmCre MyD88FL/FL mice had a greater incidence of adenocarcinoma,although both LysmCre MyD88FL/FL mice and WT mice have adenoma and atypical hyperplasia.The increase in both tumor number and size in LysmCre MyD88FL/FL mice leads to a notable increase in tumor burden(number of tumors multiplied by the average tumor size).LysmCre MyD88FL/FL mice colons tended to be shorter than WT mice colons after AOM/DSS treatment.13 days after AOM/DSS treatment,under microscope,the large intestine of LysmCre MyD88FL/FL mice showed accumulation of more inflammatory cells such as neutrophils,more destruction of crypt structure and villi were also visible.LysmCre MyD88FL/FL mice were found to be liable to be influenced by DSS and lost more weight,developed more serious diarrhea,higher bleeding scores than WT mice,these caused a greater disease activity index(DAI).qRT-PCR of large intestinal samples indicated that large intestine of LysmCre MyD88FL/FL mice had higher mRNA levels of several pro-inflammatory cytokines when compared with WT mice,such as IL6,IL1?,CCL2,TNFa;large intestine of LysmCre MyD88FL/FL mice had higher mRNA levels of several of mitogenic responsiveness about wound healing such as IL6,IL11;higher mRNA levels of Relm-?,which is produced after bacterial translocation to the intestinal mucosa and is antibacterial peptide,higher mRNA levels of MMP10,HIF-la,which are connected with angiogenesis and tissue remodeling.Consistent with tendency of large intestinal cytokines observed via qRT-PCR,serum from LysmCreMyD88FL/FL mice after AOM/DSS treatment showed higher levels of IL6 expression by ELISA,IL17A expression is also higher than in WT mice.To more deeply analyze the inflammatory microenvironment in CAC,the immune cells infiltration in colon such as CD45+ cells,CD11b+ cells,neutrophils,monocytes,eosinophils,macrophages,CD8+T cells,CD4+T cells were analyzed by flow cytometer.By FACS we can see that there is no difference in proportion between the same kind of untreated LysmCreMyD88FL/FL mice and WT mice.We found that,on innate immunity level,13 and 80 days after AOM/DSS treatment,the myeloid cells,CD45+CD1 lb+cells in colon lamina propria from LysmCre MyD88FL/FL mice were significantly increased.We found that on day 13 after AOM/DSS treatment,the first cycle of treatment,the proportion and absolute number of CDllb+GrlhlghLy6C+myeloid cells(neutrophils),in LysmCre MyD88FL/FLmice were significantly increased,the proportion of neutrophils in WT mice is 10%of CD11b+cells,but in LysmCreMyD88FL/FL mice is about 15%(80 days after AOM/DSS treatment,the proportion is increased to 30%),indicate that in the first and three cycles of AOM/DSS administration,Gr1highCD11b+myeloid cells are increased significantly,which was further supported by immunofluorescence staining of neutrophils and macrophages on tissue sections.Even after the third cycle of AOM/DSS treatment we still found that neutrophil levels of LysmCre MyD88FL/FLmice to be much higher than those of WT mice in the large intestine,which was correlated with the increased tumor burden in LysmCre MyD88FL/FL Lmice.The increased proportion of neutrophils in large intestine might be migrated from hematopoietic stem cells(Lin-Scal+c-kithi cells,LSK)in BM of LysmCre MyD88FL/FL mice,more LSKs and MP(Myeloid progenitors cells)among Lin-cells in the BM from AOM/DSS treated LysmCreMyD88FL/FL mice was observed.We found that after third cycles of DSS treatment,the proportion of CD11b+Gr1highLy6C+myeloid cells(neutrophils),in LysmCreMyD88FL/FL mice were significantly increased in large intestine.Interestingly,when we test the neutrophils in the BM,an opposite of the pattern was observed in the BM,in which the neutrophil levels of LysmCre MyD88FL/FL mice were lower than those of WT and untreated mice;the neutrophil levels of LysmCre MyD88FL/FLmice were higher than those of WT mice in the blood.We examine the levels of IL-1? mRNA of FACS-sorted large intestinal neutrophils from AOM/DSS treated LysmCreMyD88FL/FL or WT mice by qRT-PCR,and observed that the levels of IL-1? mRNA from neutrophils of LysmCreMyD88FL/FL mice were significantly upregulated.Increased neutrophils and higher levels of IL-1? mRNA make a pro-tumorigenic environment for LysmCreMyD88FL/FL Lmice.We found that although there were no significant changes in the proportion of CD8+T cells in the lamina propria between LysmCreMyD88FL/FL mice and WT mice,IFN-y expression by CD8+T cells of LysmCreMyD88FL/FL mice was significantly decreased when compared to WT mice.We did flowjo analysis,a significant increase in the percentage of Treg cells was found in the colon lamina propria of LysmCreMyD88FL/FL mice on day 80.LysmCreMyD88FL/FL mice have more CD25+IL17A+T cells in large intestine of LysmCreMyD88FL/FL mice than in WT mice by immunofluorescence,but there were no significant difference of IL-2 and IL-10 expression in large intestine by qRT-PCR between LysmCreMyD88FL/FL mice and WT mice on day 80 after AOM/DSS treatment.We found there to be less Ki67 staining of the large intestinal mucosal epithelial cell proliferation on the third day of DSS administration after AOM injection than in WT mice,but there was more epithelial cell apoptosis as indicated by TUNEL staining in LysmCreMyD88FL/FL mice than in WT mice.Under microscope,we can see most Ki67 positive cells are in the intestinal crypt,but most TUNEL positive cells are in top of the intestinal gland.We performed Western blot analysis and we found that the expression of COX-2,p-STAT3,?-catenin and CyclinDl proteins in LysmCreMyD88FL/FL mice were more higher than in WT mice after the third cycle,the expression of COX-2,p-STAT3 and cyclinDl proteins in LysmCreMyD88FL/FL mice was more higher than in WT mice by immunohistochemical staining.DNA repair genes such as Parp1,mlh1,ATM and ATR were found to show less expression in the large intestine of Lysm CreMyD88FL/FL mice than in WT mice in the early stage of CAC,suggesting that LysmCre MyD88FL/FLmice are liable to possess DNA damage after AOM/DSS treated,perhaps the increased proinflammatory cytokines rendered higher genetic instability in the epithelial cells,so LysmCre MyD88FL/FL mice are liable to have intestinal epithelial cells cancerization.We did DNA sequencing showed that ?-catenin mutation in the polyps resected from LysmCre MyD88FL/FL mice to be more common than in those from WT mice.Both untreated LysmCre MyD88FL/FL mice(n=3)and WT mice(n=5)showed no mutations.However,44%of AOM/DSS-treated LysmCre MyD88FL/FLmice(4 of 9)were found to have mutations in exon 3.No mutations were found in AOM/DSS-treated WT mice(0 of 8 mice),the detected ?-catenin mutations were aggregated at the sites encode functional residues for phosphorylation or for ubiquitination,at an incidence of 33%,so as to affect ?-catenin degradation.In line with this,we indeed found that myeloid MyD88 signaling deficiency result in more ?-catenin accumulate in cytoplasm and nuclear by immunohistological staining.Conclusion:1.Mice have more tumors and more serious inflammatory after the mice were selected deleting MyD88 in myeloid cells after AOM/DSS administration.2.After LysmCreMyD88FL/FL mice received AOM/DSS administration,neutrophils which can secret IL-? mRNA was increased in intestinal LP,Treg cells in intestinal LP were higher and IFN-? expression by CD8+T cells was inhibited.3.After received AOM/DSS administration,LysmCreMyD88FL/FL mice show less mucosal epithelial cell proliferation and more epithelial cell apoptosis,the restore function was attenuated,then intestinal inflammation continue occur;simultaneously,LysmCreMyD88FL/FL mice are difficult to repair injured genes,and have more ?-catenin mutations,so there are more tumors in LysmCreMyD88FL/FL mice.Sense:We testify firstly:Myeloid cells protect mice from colon cancer through Myd88 signal pathway by inhibiting pro-inflammatory and pro-tumor environment forming,promote injured genes restore and maintain DNA stable in intestinal epithelial cells.